Novel ectoparasite saliva proteins and apparatus to collect such proteins

ABSTRACT

The present invention is directed to a novel product and method for isolating ectoparasite saliva proteins, and a novel product and method for detecting and/or treating allergic dermatitis in an animal. The present invention includes a saliva protein collection apparatus capable of collecting ectoparasite saliva proteins substantially free of contaminating material. The present invention also relates to ectoparasite saliva proteins, nucleic acid molecules having sequences that encode such proteins, and antibodies raised against such proteins. The present invention also includes methods to obtain such proteins and to use such proteins to identify animals susceptible to or having allergic dermatitis. The present invention also includes therapeutic compositions comprising such proteins and their use to treat animals susceptible to or having allergic dermatitis.

FIELD OF THE INVENTION

[0001] The present invention relates to a novel product and method forisolating ectoparasite saliva proteins, and a novel product and methodfor detecting and/or treating allergic dermatitis in an animal.

BACKGROUND OF THE INVENTION

[0002] Bites from ectoparasites, in particular fleas, can cause ahypersensitive response in animals. In particular, hypersensitiveresponses to fleabites is manifested in a disease called flea allergydermatitis (FAD). Hypersensitivity refers to a state of alteredreactivity in which an animal, having been previously exposed to acompound, exhibits an allergic response to the compound upon subsequentexposures. Hypersensitive responses include Type I, Type II, Type IIIand Type IV hypersensitivities. Type I hypersensitivity is described asIgE-mediated hypersensitivity in which an allergen induces cross-linkageof IgE bound to Fc receptors on the surface of mast cells. Thiscross-linkage results in the degranulation of the mast cells. Type IIhypersensitivity is described as antibody-mediated cytotoxichypersensitivity in which antibodies bind to cell surface allergensresulting in cell destruction via complement activation. Type IIIhypersensitivity is described as immune complex-mediatedhypersensitivity in which allergen-antibody complexes deposit in varioustissues and induce inflammatory responses. A delayed hypersensitivereaction includes Type IV hypersensitivity which is described as acell-mediated hypersensitivity in which T lymphocytes (i.e., T cells)release cytokines that activate macrophages or cytotoxic T cells whichmediate cellular destruction.

[0003] A Type I hypersensitive response usually occurs within about 2 to30 minutes following exposure to an allergenic compound, which isusually a soluble allergen. Type II and III responses can occur fromabout 2 to 8 hours following exposure to an allergenic compound.Alternatively, in a delayed hypersensitivity response, the allergicresponse by an animal to an allergenic compound typically is manifestedfrom about 24 to about 72 hours after exposure to the compound. Duringthe 24-hour delay, mononuclear cells infiltrate the area where the agentis located. The infiltrate can include lymphocytes, monocytes,macrophages and basophils. Lymphokines (e.g., interferon-γ) are producedwhich activate monocytes or macrophages to secrete enzymes (e.g.,proteases) which cause tissue damage.

[0004] Foreign compounds that induce symptoms of immediate and/ordelayed hypersensitivity are herein referred to as allergens. The term“allergen” primarily refers to foreign compounds capable of causing anallergic response. The term can be used interchangeably with the term“antigen,” especially with respect to a foreign compound capable ofinducing symptoms of immediate and/or delayed hypersensitivity. Factorsthat influence an animal's susceptibility to an allergen can include agenetic component and/or environmental exposure to an allergen. Animalscan be de-sensitized to an allergen by repeated injections of theallergen to which an animal is hypersensitive.

[0005] FAD can have manifestations of both immediate and delayed-typehypersensitivity. Typically, an immediate hypersensitive response in ananimal susceptible to FAD includes wheal formation at the site of afleabite. Such wheals can develop into a papule with a crust,representative of delayed-type hypersensitivity. Hypersensitivereactions to fleabites can occur in genetically pre-disposed animals aswell as in animals sensitized by previous exposure to fleabites.

[0006] Effective treatment of FAD has been difficult if not impossibleto achieve. FAD afflicts about 15% of cats and dogs in flea endemicareas and the frequency is increasing each year. In a geographical area,effective flea control requires treatment of all animals. One treatmentinvestigators have proposed includes desensitization of animals usingflea allergens. However, reliable, defined preparations of fleaallergens are needed for such treatments.

[0007] Until the discovery of the novel formulations of the presentinvention, flea allergens responsible for FAD had not been clearlydefined. Whole flea antigen preparations have been used to diagnose anddesensitize animals with FAD (Benjamini et al., 1960, pp. 214-222,Experimental Parasitology, Vol. 10; Keep et al., 1967, pp. 425-426,Australian Veterinary Journal, Vol. 43; Kristensen et al., 1978, pp.414-423, Nord. Vet-Med, Vol. 30; Van Winkle, 1981, pp. 343-354, J. Amer.Animal Hosp. Assoc., Vol. 17; Haliwell et al., 1987, pp. 203-213,Veterinary Immunology and Immunopathology, Vol. 15; Greene et al., 1993,pp. 69-74, Parasite Immunology, Vol. 15); PCT Publication No. WO93/18788 by Opdebeeck et al.; and Van Winkle, pp. 343-354, 1981, J. Am.Anim. Hosp. Assoc., vol. 32. Available commercial whole flea extracts,however, are unpredictable and, therefore, have limited usefulness.

[0008] Prior investigators have suggested that products contained inflea saliva might be involved in FAD and have also suggested methods toisolate such products: Benjamini et al., 1963, pp. 143-154, ExperimentalParasitology, Vol. 13; Young et al., 1963, pp. 155-166, ExperimentalParasitology 13, Vol. 13; Michaeli et al., 1965, pp. 162-170, J.Immunol., Vol. 95; and Michaeli et al., 1996, pp. 402-406, J. Immunol.,Vol. 97. These investigators, however, have characterized the allergenicfactors of flea saliva as being haptens having molecular weights of lessthan 6 kilodaltons (kD). That they are not proteins is also supported bythe finding that they are not susceptible to degradation when exposed tostrong acids (e.g., 6 N hydrochloric acid) or heat. Some of theparticular low molecular weight allergenic factors have also beencharacterized as being a highly fluorescent aromatic fraction (Young etal., ibid.). In addition, studies by such investigators have indicatedthat in order to be allergenic, such factors need to be associated withadjuvants and/or carriers, such as collagen or portions of the membraneused to collect the oral secretions. Moreover, the methods described tocollect flea saliva factors were difficult and unpredictable.Furthermore the factors isolated by these methods were typicallycontaminated with material from the fleas, their culture medium or theskin-based membranes used to allow the fleas to feed.

[0009] Thus, there remains a need to more clearly define flea salivaallergens capable of inducing a hypersensitive response in animals. Inaddition, there remains a need to develop a method to collectsubstantially pure flea saliva allergens which provide predictable andless expensive preparations of allergens useful for desensitizinganimals subject to, or having, FAD.

SUMMARY OF THE INVENTION

[0010] The present invention relates to, in one embodiment, aformulation comprising at least one isolated ectoparasite salivaprotein, in which the ectoparasite saliva protein comprises at least aportion of an amino acid sequence, in which the portion is encoded by anucleic acid molecule capable of hybridizing under stringent conditionswith a nucleic acid molecule that encodes a flea saliva protein presentin flea saliva extract FS-1, FS-2 and/or FS-3. Preferred flea salivaproteins include fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF,fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2,fspM1, fspM2, fspN1, fspN2 and/or fspN3. In addition, the flea salivaprotein of the formulation can include at least a portion of an aminoacid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54 and/or SEQ ID NO:56.

[0011] Another embodiment of the present invention includes aformulation comprising at least one isolated ectoparasite salivaprotein, in which the ectoparasite saliva protein comprises at least aportion of an amino acid sequence, in which the portion is encoded by anucleic acid molecule capable of hybridizing under stringent conditionswith a nucleic acid molecule that encodes a flea saliva proteinrepresented as a protein peak in FIG. 2.

[0012] One aspect of the present invention includes a formulationcomprising an ectoparasite saliva product, in which the formulation,when submitted to Tris glycine SDS-PAGE, comprises a fractionationprofile as depicted in a FIG. 1B, lane 13 and/or FIG. 1B, lane 14.

[0013] Yet another embodiment of the present invention includes aformulation comprising at least one isolated ectoparasite saliva productsubstantially free of contaminating material, the formulation beingproduced by a process comprising: (a) collecting ectoparasite salivaproducts on a collection means within a saliva collection apparatuscontaining ectoparasites, the apparatus comprising (i) a housingoperatively connected to a chamber, the chamber having an ambienttemperature warmer than the housing thereby forming a temperaturedifferential between the housing and the chamber, the housing beingcapable of retaining ectoparasites, and (ii) an interface between thehousing and the chamber, the interface comprising ((a)) a means capableof collecting at least a portion of saliva products deposited byectoparasites retained in the apparatus and ((b)) a barrier meanscapable of substantially preventing contaminating material fromcontacting the collection means, in which the temperature differentialattracts ectoparasites retained in the housing to attempt to feedthrough the barrier means and collection means and, thereby, depositsaliva products on the collection means; and (b) extracting the salivaproducts from the collection means to obtain the formulation. Alsoincluded in the present invention is such an apparatus and use such anapparatus to produce formulations comprising flea saliva productssubstantially free of contaminating material.

[0014] Another aspect of the present invention includes an isolatednucleic acid molecule capable of hybridizing under stringent conditionswith a gene encoding a flea saliva protein present in flea salivaextract FS-1, FS-2 and/or FS-3, including, but not limited to fspA,fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH,fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2and/or fspN3. In particular, the nucleic acid molecule is capable ofhybridizing under stringent conditions with nucleic acid sequence SEQ IDNO: 20, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 50, SEQID NO: 52 or SEQ ID NO: 55. Also included in the present invention arerecombinant molecules and recombinant cells having a nucleic acidmolecule of the present invention.

[0015] Also included in the present invention is a method for producingat least one ectoparasite saliva protein, comprising: (a) culturing acell transformed with at least one nucleic acid capable of hybridizingunder stringent conditions with a gene encoding a flea saliva proteinpresent in flea saliva extract FS-1, FS-2, and/or FS-3 to produce theprotein; and (b) recovering the ectoparasite saliva proteins.

[0016] Another aspect of the present invention includes an antibodycapable of selectively binding to an ectoparasite saliva product, ormimetope thereof.

[0017] Yet another aspect of the present invention includes atherapeutic composition for treating allergic dermatitis comprising anyof the formulations disclosed herein. In particular, the therapeuticcomposition is useful for treating flea allergy dermatitis, mosquitoallergy dermatitis and/or Culicoides allergy dermatitis. Moreover,particular flea saliva proteins to include in a therapeutic compositioninclude at least a portion of at least one of the following flea salivaproteins: fspE, fspF, fspG1 fspG2 fspG3, fspH, fspI, fspJ1, fspJ2, fspK,fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and/or fspN3. The presentinvention also includes a method to desensitize a host animal toallergic dermatitis, comprising administering to the animal atherapeutic composition.

[0018] The present invention further relates to an assay kit for testingif an animal is susceptible to or has allergic dermatitis, the kitcomprising: (a) a formulation as disclosed herein; and (b) a means fordetermining if the animal is susceptible to or has allergic dermatitis,in which the means comprises use of the formulation to identify animalssusceptible to or having allergic dermatitis.

[0019] According to the present invention, a method can be used toidentify an animal susceptible to or having allergic dermatitis, themethod comprising: (a) administering to a site on the animal aformulation of the present invention and administering to a differentsite on the animal a control solution selected from the group consistingof positive control solutions and negative control solutions; and (b)comparing a reaction resulting from administration of the formulationwith a reaction resulting from administration of the control solution.The animal is determined to be susceptible to or to have allergicdermatitis if the reaction to the formulation is at least as large asthe reaction to the positive control solution. The animal is determinednot to be susceptible to or not to have allergic dermatitis if thereaction to the formulation is about the same size as the reaction tothe negative control solution. In particular, the method can detectimmediate hypersensitivity and/or delayed hypersensitivity.

[0020] Also according to the present invention, a method can be used toidentify an animal susceptible to or having allergic dermatitis bymeasuring the presence of antibodies indicative of allergic dermatitisin the animal, the method comprising: (a) contacting a formulation ofthe present invention with a body fluid from the animal under conditionssufficient for formation of an immunocomplex between the formulation andthe antibodies, if present, in the body fluid; and (b) determining theamount of immunocomplex formed, in which formation of the immunocomplexindicates that the animal is susceptible to or has allergic dermatitis.In particular, the method can be used to detect IgE antibodies as anindicator of immediate hypersensitivity in the animal.

[0021] The present invention also includes a method for prescribingtreatment for allergic dermatitis, comprising: (a) identifying an animalthat is susceptible to or has allergic dermatitis by an in vivo or invitro assay comprising a formulation of the present invention; and (b)prescribing a treatment comprising administering formulation of thepresent invention to the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1A illustrates the resolution of flea saliva proteins byreducing 16% Tris glycine SDS-PAGE.

[0023]FIG. 1B illustrates the resolution of flea saliva proteins, FS-1and FS-2 by reducing 16% Tris glycine SDS-PAGE.

[0024]FIG. 1C illustrates the resolution of fspN by reducing 16% Trisglycine SDS-PAGE.

[0025]FIG. 2 illustrates the resolution of flea saliva proteins usinghigh pressure liquid chromatography.

[0026]FIG. 3 illustrates the peaks obtained from reverse phase HPLCresolution of proteolytic fragments of fspH protein digested withEndoproteinase Asp-N.

[0027]FIG. 4A illustrates a cross-section of a flea saliva collectionapparatus of the present invention.

[0028]FIG. 4B illustrates a blow-out of a flea saliva collectionapparatus of the present invention.

[0029]FIG. 5 illustrates the relative size of wheals produced 15 minutesafter injection of various flea saliva protein formulations intoflea-sensitized dogs.

[0030]FIG. 6 illustrates the relative induration of wheals 6 hours afterinjection of various flea saliva protein formulations intoflea-sensitized dogs.

[0031]FIG. 7 illustrates the relative erythema of wheals 6 hours afterinjection of various flea saliva protein formulations intoflea-sensitized dogs.

[0032]FIG. 8 illustrates the relative induration of wheals 24 hoursafter injection of various flea saliva protein formulations intoflea-sensitized dogs.

[0033]FIG. 9 illustrates the relative erythema of wheals 24 hours afterinjection of various flea saliva protein formulations intoflea-sensitized dogs.

[0034]FIG. 10 depicts ELISA results measuring anti-flea saliva IgEantibodies in the sera of flea sensitized dogs.

[0035]FIGS. 11A and 11B depict ELISA results measuring anti-flea salivaIgE antibodies in the serum of a flea sensitized dog and the lackthereof in heartworm infected dogs.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention includes a novel product and method fordiagnosing and treating allergic dermatitis of animals to ectoparasites.The invention is particularly advantageous in that it provides for aunique formulation of ectoparasite saliva products sufficiently free ofcontaminants such as blood proteins, fecal material and larval culturemedium, to be useful in diagnosis and therapy of allergies caused byectoparasites. In addition, the present invention includes ectoparasitesaliva products having other activities, important, for example, in aflea's ability to feed and/or counteract a host's resistance to fleas,such as products having clotting, anti-coagulant, protease,phospholipase, prostaglandin, anti-complement, other immunosuppressant,phosphatase, apyrase, vasoactive, and/or anti-inflammatory activities.Included among flea saliva products are products, such as, but notlimited to, proteases, that are regurgitated by the flea that originatedin other organs, such as, but not limited to, the midgut.

[0037] The invention is also particularly advantageous in that itprovides an apparatus and method for reproducibly and efficientlyisolating ectoparasite saliva products substantially free ofcontaminating material.

[0038] According to the present invention, ectoparasites are externalliving parasites that attach and feed through the skin of a host animal.Ectoparasites include parasites that live on a host animal and parasitesthat attach temporarily to an animal in order to feed. Also, accordingto the present invention, ectoparasite saliva refers to the materialreleased from the mouth of an ectoparasite when the ectoparasiteattempts to feed in response to a temperature differential, such asexists in an apparatus of the present invention. Ectoparasite salivaincludes ectoparasite saliva products. Ectoparasite saliva products ofthe present invention comprise the portion of ectoparasite saliva boundto a collecting means of the present invention (described in detailbelow), herein referred to as ectoparasite saliva components. As such,ectoparasite saliva products also include the portion of ectoparasitesaliva extracted from a collecting means of the present invention,herein referred to as ectoparasite saliva extract. Included inectoparasite saliva extracts are ectoparasite saliva proteins which canbe isolated using, for example, any method described herein.Ectoparasite saliva extracts of the present invention can also includeother ectoparasite saliva products, such as, prostaglandins and otherpharmacologically active molecules.

[0039] One embodiment of the present invention is a formulation thatcontains ectoparasite saliva products that can be used to diagnoseand/or treat animals susceptible to or having (i.e., suffering from)allergic dermatitis. Preferred types of allergic dermatitis to diagnoseand/or treat using ectoparasite saliva products of the present inventioninclude flea allergy dermatitis, Culicoides allergy dermatitis andmosquito allergy dermatitis. A preferred type of allergic dermatitis todiagnose and/or treat using ectoparasite saliva products of the presentinvention is flea allergy dermatitis. As used herein, an animal that issusceptible to allergic dermatitis refers to an animal that isgenetically pre-disposed to developing allergic dermatitis and/or to ananimal that has been primed with an antigen in such a manner thatre-exposure to the antigen results in symptoms of allergy that can beperceived by, for example, observing the animal or measuring antibodyproduction by the animal to the antigen. As such, animals susceptible toallergic dermatitis can include animals having sub-clinical allergicdermatitis. Sub-clinical allergic dermatitis refers to a condition inwhich allergy symptoms cannot be detected by simply observing an animal(i.e., manifestation of the disease can include the presence ofanti-ectoparasite saliva protein antibodies within an affected animalbut no dermatitis). For example, sub-clinical allergic dermatitis can bedetected using in vivo or in vitro assays of the present invention, asdescribed in detail below. Reference to animals having allergicdermatitis includes animals that do display allergy symptoms that can bedetected by simply observing an animal and/or by using in vivo or invitro assays of the present invention, as described in detail below.

[0040] One embodiment of the present invention is a formulation thatincludes one or more isolated ectoparasite saliva proteins. According tothe present invention, an isolated protein is a protein that has beenremoved from its natural milieu. An isolated ectoparasite saliva proteincan, for example, be obtained from its natural source, be produced usingrecombinant DNA technology, or be synthesized chemically. As usedherein, an isolated ectoparasite saliva protein can be a full-lengthectoparasite saliva protein or any homologue of such a protein, such asan ectoparasite saliva protein in which amino acids have been deleted(e.g., a truncated version of the protein, such as a peptide), inserted,inverted, substituted and/or derivatized (e.g., by glycosylation,phosphorylation, acetylation, myristylation, prenylation, palmitation,amidation and/or addition of glycosylphosphatidyl inositol). A homologueof an ectoparasite saliva protein is a protein having an amino acidsequence that is sufficiently similar to a natural ectoparasite salivaprotein amino acid sequence that a nucleic acid sequence encoding thehomologue is capable of hybridizing under stringent conditions to (i.e.,with) a nucleic acid sequence encoding the natural ectoparasite salivaprotein amino acid sequence. As used herein, stringent hybridizationconditions refer to standard hybridization conditions under whichnucleic acid molecules, including oligonucleotides, are used to identifysimilar nucleic acid molecules. Such standard conditions are disclosed,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Labs Press, 1989. The minimal size of a proteinhomologue of the present invention is a size sufficient to be encoded bya nucleic acid molecule capable of forming a stable hybrid with thecomplementary sequence of a nucleic acid molecule encoding thecorresponding natural protein. As such, the size of the nucleic acidmolecule encoding such a protein homologue is dependent on nucleic acidcomposition and percent homology between the nucleic acid molecule andcomplementary sequence as well as upon hybridization conditions per se(e.g., temperature, salt concentration, and formamide concentration).The minimal size of such nucleic acid molecules is typically at leastabout 12 to about 15 nucleotides in length if the nucleic acid moleculesare GC-rich and at least about 15 to about 17 bases in length if theyare AT-rich. As such, the minimal size of a nucleic acid molecule usedto encode an ectoparasite saliva protein homologue of the presentinvention is from about 12 to about 18 nucleotides in length. There isno limit, other than a practical limit, on the maximal size of such anucleic acid molecule in that the nucleic acid molecule can include aportion of a gene, an entire gene, or multiple genes, or portionsthereof. Similarly, the minimal size of an ectoparasite saliva proteinhomologue of the present invention is from about 4 to about 6 aminoacids in length, with preferred sizes depending on whether afull-length, multivalent (i.e., fusion protein having more than onedomain each of which has a function), or functional portions of suchproteins are desired.

[0041] Ectoparasite saliva protein homologues can be the result ofallelic variation of a natural gene encoding an ectoparasite salivaprotein. A natural gene refers to the form of the gene found most oftenin nature. Ectoparasite saliva protein homologues can be produced usingtechniques known in the art including, but not limited to, directmodifications to a gene encoding a protein using, for example, classicor recombinant DNA techniques to effect random or targeted mutagenesis.

[0042] Preferred ectoparasite saliva proteins of the present invention,including homologues thereof, are capable of detecting and/or treatingallergic dermatitis resulting from the bites of ectoparasites. Apreferred ectoparasite saliva protein homologue includes at least oneepitope capable of eliciting a hypersensitive response to the naturalectoparasite saliva protein counterpart. An ectoparasite saliva proteinhomologue can also include an epitope capable of hyposensitizing ananimal to the natural form of the protein. The ability of anectoparasite saliva protein homologue to detect and/or treat (i.e.,immunomodulate or regulate by, for example, desensitizing) thehypersensitivity of an animal susceptible to or having allergicdermatitis, can be tested using techniques known to those skilled in theart. Such techniques include skin tests and immunoabsorbent assays asdescribed in detail below. Additional preferred ectoparasite salivaproteins of the present invention have other activities that includeactivities important for feeding and survival of the ectoparasite.

[0043] In one embodiment, a formulation of the present invention cancomprise a protein having at least a portion of an isolated ectoparasitesaliva protein. According to the present invention, “at least a portionof an ectoparasite saliva protein” refers to a portion of anectoparasite saliva protein encoded by a nucleic acid molecule that iscapable of hybridizing, under stringent conditions, with a nucleic acidencoding a full-length ectoparasite saliva protein of the presentinvention. Preferred portions of ectoparasite saliva proteins are usefulfor detecting and/or treating allergic dermatitis resulting from thebites of ectoparasites. Additional preferred portions have activitiesimportant for flea feeding and survival. Suitable sizes for portions ofan ectoparasite saliva protein of the present invention are as disclosedfor saliva protein homologues of the present invention.

[0044] As will be apparent to one of skill in the art, the presentinvention is intended to apply to all ectoparasites. A formulation ofthe present invention can include saliva products from anyectoparasites. A preferred ectoparasite of the present invention fromwhich to isolate saliva products (including proteins), and/or from whichto identify proteins that can then be produced recombinantly orsynthetically, include arachnids, insects and leeches. More preferredectoparasites from which to obtain saliva products include fleas; ticks,including both hard ticks of the family Ixodidae (e.g., Ixodes andAmblyomma) and soft ticks of the family Argasidae (e.g., Ornithodoros,such as o. parkeri and o. turicata); flies, such as midges (e.g., Culicides), mosquitos, sand flies, black flies, horse flies, horn flies, deerflies, tsetse flies, stable flies, myiasis-causing flies and bitinggnats; ants; spiders, lice; mites; and true bugs, such as bed bugs andkissing bugs, including those carrying Chagas disease. Even morepreferred ectoparasite saliva products include those from fleas,mosquitos, midges, sandflies, blackflies, ticks and Rhodnius, withrroducts from fleas, mosquitos and Culicoides being even more preferred.

[0045] A particularly preferred formulation of the present inventionincludes flea saliva proteins. Preferred flea saliva products includethose from Ctenocephalides, Xenopsylla, Pulex, Tunga, Nosopsyllus,Diamanus, Ctopsyllus and Echidnophaga fleas, with saliva products fromCtenocephalides canis and Ctenocephalides felis fleas being even morepreferred. For the purposes of illustration, many of the followingembodiments discuss flea saliva proteins. Such discussion of flea salivaproteins is not intended, in any way, to limit the scope of the presentinvention.

[0046] In one embodiment, a formulation of the present invention issubstantially free from contaminating material. Contaminating materialcan include, for example, ectoparasite fecal material, blood proteinsfrom previous meals taken by an ectoparasite (e.g., fetuin, ferritin,albumin, hemoglobin and other large blood proteins), ectoparasitecuticular debris, and ectoparasite larval culture medium (e.g., blood,mouse food and sand). As used herein, a formulation that issubstantially free of contaminants is a formulation that without furtherpurification can be used as a diagnostic or therapeutic agent withoutcausing undesired side effects. Preferably, a formulation substantiallyfree from contaminating material comprises less than about 50 percentcontaminating material, more preferably less than about 10 percentcontaminating material, and even more preferably less than about 5percent contaminating material. As such, a formulation of the presentinvention preferably comprises at least about 50 percent flea salivaproducts, more preferably at least about 90 percent flea salivaproducts, and even more preferably at least about 95 percent flea salivaproducts. A formulation of the present invention substantially free ofcontaminating material can include a formulation not having any bloodcontaminants or flea midgut contents. A formulation substantially freeof contaminating material can be obtained using a saliva collectionapparatus of the present invention as described in detail below.

[0047] A formulation that is substantially free of contaminatingmaterial can be identified by typical methods known to those of skill inthe art. For example, the presence of contaminants can be identified by:(1) overloading and resolving a formulation by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE); (2) resolving aformulation by a variety of chromatography techniques; (3) screening aformulation with antibodies capable of binding to specific contaminantsusing, for example, immunoblot or enzyme-linked immunoassay techniques;(4) resolving a formulation by capillary electrophoresis; or (5)screening a formulation using an assay to detect hemoglobin.

[0048] One embodiment of a formulation of the present invention includesat least one or more flea saliva proteins having molecular weightsranging from about 6 kD to about 65 kD as determined by Tris-glycineSDS-PAGE, preferably using a 14% polyacrylamide gel and resolved usingmethods standard in the art. A preferred formulation includes one ormore flea saliva proteins having molecular weights ranging from about 6kD to about 55 kD. A more preferred formulation includes one or moreproteins having elution (or migration) patterns as shown in FIG. 1.

[0049] In another embodiment, a formulation of the present inventionincludes at least one or more flea saliva proteins having molecularweights ranging from about 40 kD to about 300 kD as determined byTris-glycine SDS-PAGE and resolved using methods standard in the art.Greater than 50% of the flea saliva proteins contained in such aformulation have a molecular weights ranging from about 40 kD to about55 kD, and appear to be similar to fspN. A more preferred formulationincludes one or more proteins having alution (or migration) patterns asshown in FIG. 1.

[0050] In another embodiment, a formulation of the present inventionincludes one or more flea saliva proteins having basic isoelectricpoints, or pI values. An isoelectric pH, or pI, value refers to the pHvalue at which a molecule has no net electric charge and fails to movein an electric field. A preferred formulation of the present inventionincludes proteins having a pI value of at least about pI 8.5, and morepreferably of at least about pI 9.0. Flea saliva protein fspH, forexample, has pI values ranging from about pI 8.5 to about pI 9.6, whichmay represent heterogeneity in the proteins due to allelic variation inthe flea population from which the flea saliva proteins were collected.

[0051] In yet another embodiment, a formulation of the present inventionincludes at least a portion of one or more flea saliva products elutedfrom a collection means of the present invention. Examples of suchformulations include flea extracts FS-1, FS-2, and FS-3. The FS-1, FS-2and FS-3 fleas saliva extracts are produced according to the methoddescribed in detail in Example 2. According to the present invention,the terms FS-1 flea saliva extract, FS-2 flea saliva extract or FS-3flea saliva extract can be used interchangeably with the terms FS-1 fleasaliva product mixture, FS-2 flea saliva product mixture or FS-3 fleasaliva product mixture, respectively.

[0052] An FS-1 flea saliva extract includes a mixture of proteins (a)that, when submitted to reducing 16% Tris glycine SDS-PAGE, migrate asbands as are shown in FIG. 1B, lane 13; and (b) that, when submitted toreverse phase high pressure liquid chromatography (HPLC), migrate aspeaks as are shown in FIG. 2. The peaks in FIG. 2 are obtained when theproteins included in FS-1 are collected using a saliva collectionapparatus of the present invention as described in detail below, andfurther resolved into protein peaks by passing the collected proteinsover a C4 HPLC column using 5% to 63% acetonitrile or 5.6% to 70%Solvent B at a flow rate of 0.8 milliliters per minute, in which SolventA is about 0.1% TFA in water and Solvent B is about 0.085% TFA in 90%acetonitrile. Referring to FIG. 2, the peaks are referred to anddepicted as peak A, peak B, peak C, peak D, peak E, peak F, peak G, peakH, peak I, peak J, peak K, peak L, peak M and peak N. Flea salivaproteins (or protein fragments) contained within such peaks are referredto as fspA, fspB, fspC, fspD, fspE, fspF, fspG, fspH, fspI, fspJ, fspK,fspL, fspM and fspN. The peaks refer to the regions marked in FIG. 2 andit is to be noted that a peak does not necessarily contain just oneprotein (or protein fragment). Further resolution of proteins containedwithin the above-referenced peaks by, for example, amino acid sequencingor SDS-PAGE gel electrophoresis, has indicated that fspc includes atleast two proteins referred to as fspC1 and fspC2, fspD includes atleast two proteins referred to as fspD1 and fspD2, fspG includes atleast three proteins referred to as fspG1, fspG2 and fspG3, fspJincludes at least two proteins referred to as fspJ1 and fspJ2, fspLincludes at least two proteins referred to as fspL1 and fspL2, fspMincludes at least two proteins referred to as fspM1 and fspM2, and fspNincludes at least three proteins and/or protein fragments referred to asfspN1, fspN2 and fspN3. At least partial amino acid sequences have beenobtained for a number of the flea saliva proteins as represented by SEQID NO: 1 (a partial N- (amino-) terminal amino acid sequence of fspA),SEQ ID NO: 2 (an amino acid sequence, beginning at the N-terminus, thatrepresents most of the fspH protein), SEQ ID NO: 3 (a partial N-terminalamino acid sequence of an Endoproteinase Asp-N fragment of fspH, denotedfspHe), SEQ ID NO: 4 (a partial N-terminal amino acid sequence of anEndoproteinase Asp-N fragment of fspH, denoted fspHh), SEQ ID NO: 5 (apartial N-terminal amino acid sequence of an Endoproteinase Asp-Nfragment of fspH, denoted fspHj, which also represents a partialN-terminal amino acid sequence of fspH), SEQ ID NO: 6 (a partialN-terminal amino acid sequence of fspI), SEQ ID NO: 7 (a partialN-terminal amino acid sequence of fspJ1), SEQ ID NO: 8 (a partialN-terminal amino acid sequence of fspJ2), SEQ ID NO: 9 (a partialN-terminal amino acid sequence of fspL1), SEQ ID NO: 10 (a partialN-terminal amino acid sequence of fspL2), SEQ ID NO: 11 (a partialN-terminal amino acid sequence of fspN1), SEQ ID NO: 12 (a partialN-terminal amino acid sequence of fspN2), SEQ ID NO: 13 (a partialN-terminal amino acid sequence of fspN3), SEQ ID NO: 14 (a partialN-terminal amino acid sequence of fspH), SEQ ID NO: 25 (a translation ofthe nucleic acid sequence represented by SEQ ID NO: 24, corresponding tofspI), SEQ ID NO: 26 (an apparent full-length translation product offspI), SEQ ID NO: 27 (a partial N-terminal amino acid sequence of fspB),SEQ ID NO: 28 (a partial N-terminal amino acid sequence of fspG1), SEQID NO: 29 (a partial N-terminal amino acid sequence of fspG2), SEQ IDNO: 30 (a partial N-terminal amino acid sequence of fspG3), SEQ ID NO:31 (a partial N-terminal amino acid sequence of an Endoproteinase Asp-Nfragment of fspN, denoted fspN(100-101)), SEQ ID NO: 33 (a translationproduct, named PfspH₈₀, of the partial nucleic acid sequencecorresponding to fspH, named nfspH₂₄₂, denoted SEQ ID NO: 32), SEQ IDNO: 35 (a translation product, named PfspI₁₅₅, of the partial nucleicacid sequence corresponding to fspI, named nfspI₅₉₁, denoted SEQ ID NO:34), SEQ ID NO: 51 (a translation product, named PfspN(A)₁₇₂, of thepartial nucleic acid sequence of a fspN protein, named nfspN(A)₆₄₆,denoted SEQ ID NO: 50), SEQ ID NO: 53 (a translation product, namedPfspN(B)₁₅₃, of the partial nucleic acid sequence of a fspN proteinnamed nfspN(B)₆₁₂, denoted SEQ ID NO: 52), SEQ ID NO: 54 (a partialapparent N-terminal amino acid sequence, named PfspN(A)₅₆, of a fspNprotein named PfspN(A)), and SEQ ID NO: 56 (an apparent full-lengthtranslation product, named PfspN(A)₃₉₈, of the apparent complete nucleicacid sequence of fspN3, named nfspN(A)₁₁₉₇, denoted SEQ ID NO: 55). Thedetails of how each protein was characterized is described in Examples 2and 3.

[0053] An FS-2 flea saliva extract includes a mixture of proteins that,when submitted to reducing 16% Tris glycine SDS-PAGE, migrate as bandsas are shown in FIG. 1B, lanes 14 and 15.

[0054] It is within the scope of the present invention that additionalflea saliva products of interest remain on a collection means followingthe elution protocols to obtain FS-1, FS-2 and FS-3 flea salivaextracts. It is also within the scope of the invention that aformulation of the present invention can include flea saliva productsremoved from a collection means by eluting using other techniques, forexample, by using higher concentrations of eluants.

[0055] In another embodiment, a formulation of the present inventionincludes at least a portion of an ectoparasite saliva protein homologuepreferably having at least about 50 percent, more preferably at leastabout 75 percent, and even more preferably at least about 85 percentamino acid homology (identity within comparable regions) with at least aportion of at least one product contained in the saliva extracts FS-1,FS-2 or FS-3. Preferred homologues include at least a portion of anectoparasite saliva product having at least about 50 percent, morepreferably at least about 75 percent, and even more preferably at leastabout 85 percent amino acid homology with at least a portion of one ormore of the proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF,fspG1 fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1,fspM2, fspN1, fspN2 and fspN3. As such, also included are proteinshaving at least a portion of one of the following amino acid sequences:SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ IDNO: 30, SEQ ID NO: 31 SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 51, SEQID NO: 53, SEQ ID NO: 54 and/or SEQ ID NO: 56.

[0056] In a preferred embodiment, a formulation of the present inventionincludes at least a portion of an ectoparasite saliva product homologueof the present invention that is encoded by a nucleic acid moleculehaving at least about 50 percent, more preferably at least about 75percent, and even more preferably at least about 85 percent homologywith a nucleic acid molecule encoding at least a portion of a productcontained in the saliva extracts FS-1, FS-2 or FS-3. A preferredectoparasite saliva product homologue is encoded by a nucleic acidmolecule having at least about 50 percent, more preferably at leastabout 75 percent, and even more preferably at least about 85 percent,homology with a nucleic acid molecule encoding at least a portion of oneor more of the proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE,fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2,fspM1, fspM2, fspN1, fspN2 and fspN3.

[0057] In yet another embodiment, a formulation of the present inventionincludes a protein which, when digested with Endoproteinase Asp-N,generates proteolytic fragments that, when subjected to reverse phaseHPLC, migrate with peaks as depicted in FIG. 3. The reverse phase HPLCwas performed using the methods disclosed by Stone et al., EnzymaticDigestion of Proteins and HPLC Peptide Isolation, in A Practical Guideto Protein and Peptide Purification for Microsequencing, P T Matsudairaed., Academic Press, San Diego, Calif. (i.e., Narrowbore procedure:vydac C18 reverse-phase, 300 A, 5 μm support; flow rate of 0.2 ml/min;Solvent A being 0.6% TFA in water and Solvent B being 0.052% TFA in 80%acetonitrile in water; the sample was injected at 2% B; the gradientafter a hold at 2% B was 2-37.5% B over 60 min., 37.5%-75% B over 30min., 75%-98% B over 15 min.; and detection at 214 nm). An example ofsuch a protein is fspH, which also has the characteristics of amolecular weight of about 8613±6 daltons when determined by ESMS. Aparticularly preferred formulation of the present invention includes afspH protein having the amino acid sequence represented by SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO:33.

[0058] In a preferred embodiment, a formulation of the present inventioncan include at least one isolated protein having (i.e., including) atleast a portion of the amino acid sequence (using the standard oneletter amino acid code): Y G K Q Y S E K G G R G Q R H Q I L K K G K Q YS           S K       I     L   D   L S R

[0059] (SEQ ID NO: 1; representing a partial N-terminal sequence offspA).

[0060] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: D R R V S K T C Q S G G K I Q S E X Q V V I K S G Q H/Y I L EN Y X S D G R N N N N P C H L F C N R E C R S G N G C C G N C G R T R PD S K H C Y C E A P Y S

[0061] (SEQ ID NO: 2, representing the N-terminal nearly completesequence of fspH).

[0062] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: D R R V S K T X Q S G G K I Q S E X Q V V I K S G Q H/Y I L EN Y X S D G R

[0063] (SEQ ID NO: 14, representing a partial N-terminal sequence offspH).

[0064] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0065] D S K H C Y C E A P Y S

[0066] (SEQ ID NO: 3; representing a partial N-terminal sequence offspHe)

[0067] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: D G R N N N N P C H L F C M R E C R S G N G G C G N G G R T RP D S K H C

[0068] (SEQ ID NO: 4; representing a partial N-terminal sequence offspHh).

[0069] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0070] D R R V S K T C Q S G

[0071] (SEQ ID NO: 5; representing a partial N-terminal sequence offspHj).

[0072] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: E D I W K V N K K X T S G G K N Q D R K L D Q I I Q K G Q Q VX X Q N X X K

[0073] (SEQ ID NO: 6; representing a partial N-terminal sequence offspI).

[0074] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0075] N S H E P G N T R K I R E V M D K L R K Q H P

[0076] (SEQ ID NO: 7; representing a partial N-terminal sequence offspJ1)

[0077] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0078] E I K R N S H E P G N T R K I R E V M D K L R K Q H P

[0079] (SEQ ID NO: 8; representing a partial N-terminal sequence offspJ2).

[0080] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: N D K E P G N T R K I R E V M D K L R K Q A Q P R T D G Q R PK T X I M

[0081] (SEQ ID NO: 9; representing a partial N-terminal sequence offspL1).

[0082] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0083] X L X R N D K E P G N T R K I R E V M D K

[0084] (SEQ ID NO: 10; representing a partial N-terminal sequence offspL2).

[0085] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0086] N D E L K F V F V M A K

[0087] (SEQ ID NO: 11; representing a partial N-terminal sequence offspN1).

[0088] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0089] X D E L K F V F V M A K G P S X Q A X D Y P C

[0090] (SEQ ID NO: 12; representing a partial N-terminal sequence offspN2). Note that although fspN1 and fspN2 appear to have similar, ifnot identical, partial N-terminal sequences, the two proteins migratedifferently when submitted to Tris glycine SDS-PAGE, suggesting thatthey are different proteins, possibly due to a carboxyl-terminaltruncation of one of the proteins and/or post-translation modification,cuch as glycosylation.

[0091] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0092] E L K F V F A T A R G M S H T P C D Y P

[0093] (SEQ ID NO: 13; representing a partial N-terminal sequence offspN3).

[0094] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: S G K Q Y S E X G K Q               S

[0095] (SEQ ID NO: 27; representing a partial N-terminal amino acidsequence of fspB).

[0096] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0097] D R R V S K

[0098] (SEQ ID NO: 28; representing a partial N-terminal amino acidsequence of fspG1).

[0099] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0100] S K M V T E K X K S G G N N P S T K E V S I P

[0101] (SEQ ID NO: 29; representing a partial N-terminal amino acidsequence of fspG2).

[0102] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0103] E V S I P S G K L T I E D F X I G N H Q

[0104] (SEQ ID NO: 30; representing a partial N-terminal amino acidsequence of fspG3).

[0105] A formulation of the, present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: F S L C V L Y Q I V V A D R R V S K T C Q S G G K I Q S E E/XQ V V I K S G Q H/Y I L E N Y X S D G R N N N N P C H L F C M R E C R SG N G G C G N G G R T R P D S

[0106] (SEQ ID NO: 33; representing a translation product of the nucleicacid sequence represented by SEQ ID NO: 32, corresponding to fspH).

[0107] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: L T S G G K N Q D R K L D Q I I Q K G Q Q V K I Q N I C K L IR D K P H T N Q E K E K C M K F C T K N V C K C Y R C A C D G N I C Y CS R P S N L G P D W K V N E R I E R L P I T K I L V S G N S S I S T T IT N S K Y F E T K N S E T N E D S K S K K H S K E K C R G G N D R G C DG N V L L L S T K K

[0108] (SEQ ID NO: 25; representing a translation of the nucleic acidsequence represented by SEQ ID NO: 24, corresponding to fspI).

[0109] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: E D I W K V N K K L T S G G K N Q D R K L D Q I I Q K G Q Q VK I Q N I C K L I R D K P H T N Q E K E K C M K F C T K N V C K G Y R GA C D G N I C Y C S R P S N L G P D W K V N E R I E R L P I T K I L V SG N S S I S T T I T N S K Y F E T K N S E T N E D S K S K K H S K E K CR G G N D R G C D G N V L L L S T K K

[0110] (SEQ ID NO: 26; representing an apparent full-length translationproduct of fspI).

[0111] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: W K V N K K L T S G G K N Q D R K L D Q I I Q K G Q Q V K I QN I C K L I R D K P H T N Q E K E K C M K F C T K N V C K G Y R G A C DG N I C Y C S R P S N L G P D W K V N E R I E R L P I T K I L V S G N SS I S T T I T N S K Y F E T K N S E T N E D S K S K K H S K E K C R G GN D R G C D G N V L L L S T K K

[0112] (SEQ ID NO: 35; representing a translation product of the nucleicacid sequence represented by SEQ ID NO: 34, corresponding to fspI).

[0113] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence:

[0114] D I E N I K K G E G Q P G A P G G K E N N L S V L

[0115] (SEQ ID NO: 31; representing a partial N-terminal amino acidsequence of an Endoproteinase Asp-N fragment of fspN, namedPfspN(100-101)).

[0116] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: A R A R S V G S M K N K L K S F S E K Y V W A A L T S N D N LR K M S G G R M I N D I L N D I D N I K K G N G Q P N A P G K T E N K LS V S D R S S R Y L S S I R F S L F R P R Y K I E N Q D L E P S S L Y PG Q G A L H V I E L H K D K N Q W N V K T L Y R N N D Q Q E L K P M K LA K C G D T C S Y E T F K S T L Q S Y N M D K T A H D K L C K S S

[0117] (SEQ ID NO: 51; representing a translation product of the nucleicacid sequence represented by SEQ ID NO: 50, corresponding to a fspNprotein). Comparison of amino acid sequence SEQ ID NO: 51 with aminoacid sequences reported in GenBank indicates that SEQ ID NO: 51 is about28% identical with human prostatic acid phosphatase.

[0118] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: G T R K N E L K S F S E E Y L W R A L T S N E N L R K M S G GR M I N D I L N D I D S I K E E R D N R V L L E K Q E I K L S M L T V PQ A I L A A F V S A F A P K G T K I E N Q D L G P S S L Y P G Q G A L HV I E L H K D N N Q W S V K V L Y R N N D K M E L E P M K L P S C D D KC P C E L L N Q L Y N P M I

[0119] (SEQ ID NO: 53; representing a translation product of the nucleicacid sequence represented by SEQ ID NO: 52, corresponding to a fspNprotein). Comparison of amino acid sequence SEQ ID NO: 53 with aminoacid sequences reported in GenBank indicates that SEQ ID NO: 53 is about30% identical with human prostatic acid phosphatase.

[0120] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence: M W R L L L V I S S A L I I Q N V N A E L K F V F A T A T R YV S H T P S P C D P G G P K I T N K P G D F Q R V

[0121] (SEQ ID NO: 54; representing a partial N-terminal amino acidsequence of a fspN protein).

[0122] A formulation of the present invention can also include at leastone isolated protein having at least a portion of the amino acidsequence SEQ ID NO: 56, representing an apparent full-length translationproduct of the nucleic acid sequence represented by SEQ ID NO: 55,apparently corresponding to fspN3 protein. Comparison of amino acidsequence SEQ ID NO: 56 with amino acid sequences reported in GenBankindicates that SEQ ID NO: 56 is about 30% identical with human prostaticacid phosphatase.

[0123] It is to be appreciated that ectoparasite saliva proteins of thepresent invention include, but are not limited to, full-length proteins,hybrid proteins, fusion proteins, multivalent proteins, and proteinsthat are truncated homologues of, or are proteolytic products of, atleast a portion of a protein contained in the saliva extracts FS-1, FS-2or FS-3; and preferably at least a portion of saliva protein fspA, fspB,fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI,fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and/orfspN3. As such, also included are proteins having at least a portion ofone of the following amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54and/or SEQ ID NO: 56. As used herein, the term hybrid protein refers toa single protein produced from two different proteins.

[0124] The foregoing SEQ ID NO's represent amino acid sequences deducedaccording to methods disclosed in the Examples. It should be noted thatsince amino acid sequencing technology is not entirely error-free, theforegoing SEQ ID NO's, at best, represent an apparent amino acidsequence of the ectoparasite saliva proteins of the present invention.In addition, the variation seen in the foregoing SEQ ID NO's can also bedue, at least in part, to allelic variation since the proteins beingsequenced were derived from populations of fleas.

[0125] According to the present invention, a formulation of the presentinvention can include flea saliva proteins that have undergonepost-translational modification. Such modification can include, forexample, glycosylation. Glycosylation can include addition of N-linkedand/or O-linked oligosaccharides. It is to be appreciated thatpost-translational modification of a protein of the present inventioncan contribute to an epitope's ability to induce an allergic responseagainst the protein in an immediate or delayed hypersensitivityresponse.

[0126] Another embodiment of the present invention is an isolatednucleic acid molecule capable of hybridizing, under stringentconditions, with an ectoparasite saliva protein gene encoding anectoparasite saliva protein of the present invention. In accordance withthe present invention, an isolated nucleic acid molecule is a nucleicacid molecule that has been removed from its natural milieu (i.e., thathas been subject to human manipulation). As such, “isolated” does notreflect the extent to which the nucleic acid molecule has been purified.An isolated nucleic acid molecule can include DNA, RNA, or derivativesof either DNA or RNA.

[0127] An isolated nucleic acid molecule of the present invention can beobtained from its natural source either as an entire (i.e., complete)gene or a portion thereof capable of forming a stable hybrid with thatgene. As used herein, the phrase “at least a portion of” an entityrefers to an amount of the entity that is at least sufficient to havethe functional aspects of that entity. For example, at least a portionof a nucleic acid sequence, as used herein, is an amount of a nucleicacid sequence capable of forming a stable hybrid with the correspondinggene under stringent hybridization conditions. An isolated nucleic acidmolecule of the present invention can also be produced using recombinantDNA technology (e.g., polymerase chain reaction (PCR) amplification,cloning) or chemical synthesis. Isolated ectoparasite saliva proteinnucleic acid molecules include natural nucleic acid molecules andhomologues thereof, including, but not limited to, natural allelicvariants and modified nucleic acid molecules in which nucleotides havebeen inserted, deleted, substituted, and/or inverted in such a mannerthat such modifications do not substantially interfere with the nucleicacid molecule's ability to encode an ectoparasite saliva protein of thepresent invention or to form stable hybrids under stringent conditionswith natural nucleic acid molecule isolates.

[0128] An isolated nucleic acid molecule of the present invention caninclude a nucleic acid sequence that encodes at least one ectoparasitesaliva protein of the present invention, examples of such proteins beingdisclosed herein. Although the phrase “nucleic acid molecule” primarilyrefers to the physical nucleic acid molecule and the phrase “nucleicacid sequence” primarily refers to the sequence of nucleotides on thenucleic acid molecule, the two phrases can be used interchangeably,especially with respect to a nucleic acid molecule, or a nucleic acidsequence, being capable of encoding an ectoparasite saliva protein. Asheretofore disclosed, ectoparasite saliva proteins of the presentinvention include, but are not limited to, proteins having full-lengthectoparasite saliva protein coding regions, portions thereof, and otherectoparasite saliva protein homologues.

[0129] It is to be appreciated that an ectoparasite saliva protein ofthe present invention can be encoded by a full-length nucleic acidsequence which encodes a polyprotein. The polyprotein can bepost-translationally processed into multiple proteins which are found insaliva. As used herein, an ectoparasite saliva protein gene includes allnucleic acid sequences related to a natural ectoparasite saliva proteingene such as regulatory regions that control production of anectoparasite saliva protein encoded by that gene (such as, but notlimited to, transcription, translation or post-translation controlregions) as well as the coding region itself. A nucleic acid molecule ofthe present invention can be an isolated natural ectoparasite salivaprotein nucleic acid molecule or a homologue thereof. A nucleic acidmolecule of the present invention can include one or more regulatoryregions, full-length or partial coding regions, or combinations thereof.The minimal size of an ectoparasite saliva protein nucleic acid moleculeof the present invention is the minimal size capable of forming a stablehybrid under stringent hybridization conditions with a correspondingnatural gene.

[0130] An ectoparasite saliva protein nucleic acid molecule homologuecan be produced using a rumber of methods known to those skilled in theart (see, for example, Sambrook et al., ibid.). For example, nucleicacid molecules can be modified using a variety of techniques including,but not limited to, classic mutagenesis techniques and recombinant DNAtechniques, such as site-directed mutagenesis, chemical treatment of anucleic acid molecule to induce mutations, restriction enzyme cleavageof a nucleic acid fragment, ligation of nucleic acid fragments,polymerase chain reaction (PCR) amplification and/or mutagenesis ofselected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected from a mixture of modified nucleicacids by screening for the function of the protein encoded by thenucleic acid (e.g., the ability of a homologue to elicit an allergicresponse in animals having allergic dermatitis or the ability of ahomologue to act as an anti-coagulant) and/or by hybridization withisolated ectoparasite saliva protein nucleic acids under stringentconditions.

[0131] One embodiment of the present invention is an ectoparasite salivaprotein nucleic acid molecule capable of encoding at least a portion ofa flea saliva product, or a homologue thereof (e.g., saliva products ofother ectoparasites), contained in the saliva extracts FS-1, FS-2 orFS-3, wherein FS-1, when submitted to HPLC, resolves into peak A, peakB, peak C, peak D, peak E, peak F, peak G, peak H, peak I, peak J, peakK, peak L, peak M and/or peak N. A preferred nucleic acid molecule iscapable of encoding at least a portion of one or more of the proteinsfspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3,fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2and fspN3, or homologues thereof. As such, preferred nucleic acidmolecules include, but are not limited, nucleic acid molecules thatencode proteins having at least a portion of one or more of thefollowing amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54 and/or SEQ ID NO:56, or homologues thereof.

[0132] A preferred nucleic acid molecule of the present invention iscapable of hybridizing under stringent conditions to a nucleic acid thatencodes at least a portion of a flea saliva product, or a homologuethereof (e.g., saliva products of other ectoparasites), contained in thesaliva extracts FS-1, FS-2 or FS-3. Also preferred is an ectoparasitesaliva protein nucleic acid molecule that includes a nucleic acidsequence having at least about 65 percent, preferably at least about 75percent, more preferably at least about 85 percent, and even morepreferably at least about 95 percent homology with the correspondingregion(s) of the nucleic acid sequence encoding at least a portion of aflea saliva product, or a homologue thereof (e.g., saliva products ofother ectoparasites), contained in the saliva extracts FS-1, FS-2 orFS-3. A particularly preferred nucleic acid sequence is a nucleic acidsequence having at least about 65 percent, preferably at least about 75percent, more preferably at least about 85 percent, and even morepreferably at least about 95 percent homology with a nucleic acidsequence encoding at least a portion of one or more of the proteinsfspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3,fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2and fspN3. As such, also preferred are nucleic acid molecules having atleast about 65 percent, preferably at least about 75 percent, morepreferably at least about 85 percent, and even more preferably at leastabout 95 percent homology with a nucleic acid sequence encoding at leasta portion of one or more of the following amino acid sequences: SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 51, SEQ ID NO:53, SEQ ID NO: 54 and/or SEQ ID NO: 56.

[0133] Such nucleic acid molecules can be a full-length gene and/or anucleic acid molecule encoding a full-length protein, a hybrid protein,a fusion protein, a multivalent protein or a truncation fragment. Morepreferred nucleic acid molecules of the present invention compriseisolated nucleic acid molecules having a nucleic acid sequence asrepresented by SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 50, SEQ ID NO: 52 or SEQ ID NO: 55. SEQ ID NO: 20, anucleic acid sequence that includes about 60 nucleotides of the apparentgene encoding flea saliva protein fspH, includes about 25 percent of thecoding region of fspH. SEQ ID NO: 24, a nucleic acid sequence thatincludes about 573 nucleotides of the apparent gene encoding flea salivaprotein fspI, encodes a protein of about 149 amino acids, represented bySEQ ID NO: 25. The entire translation product of fspI is apparentlyabout 158 amino acids and is denoted SEQ ID NO: 26. SEQ ID NO: 32, a 242bp nucleic acid sequence of the apparent gene encoding flea salivaprotein fspH, encodes a protein of about 80 amino acids, which isdenoted SEQ ID NO: 33. SEQ ID NO: 34, a 591 bp nucleic acid sequence ofthe apparent gene encoding flea saliva protein fspI, encodes a proteinof about 155 amino acids, which is denoted SEQ ID NO: 35. SEQ ID NO: 50,a 646 bp nucleic acid sequence of the apparent gene encoding a fspN fleasaliva protein, encodes a protein of about 172 amino acids, which isdenoted SEQ ID NO: 51. SEQ ID NO: 52, a 612 bp nucleic acid sequence ofthe apparent gene encoding a fspN flea saliva protein, encodes a proteinof about 153 amino acids, which is denoted SEQ ID NO: 53. SEQ ID NO: 55,a 1197 bp nucleic acid sequence of the apparent gene encoding fspN3,encodes a protein of about 398 amino acids, which is denoted SEQ ID NO:56.

[0134] Knowing a nucleic acid molecule of an ectoparasite saliva proteinof the present invention allows one skilled in the art to make copies ofthat nucleic acid molecule as well as to obtain a nucleic acid moleculeincluding additional portions of ectoparasite saliva protein-encodinggenes (e.g., nucleic acid molecules that include the translation startsite and/or transcription and/or translation control regions), and/orectoparasite saliva protein nucleic acid molecule homologues. Knowing aportion of an amino acid sequence of an ectoparasite saliva protein ofthe present invention allows one skilled in the art to clone nucleicacid sequences encoding such an ectoparasite saliva protein. Inaddition, a desired ectoparasite saliva protein nucleic acid moleculecan be obtained in a variety of ways including screening appropriateexpression libraries with antibodies which bind to ectoparasite salivaproteins of the present invention; traditional cloning techniques usingoligonucleotide probes of the present invention to screen appropriatelibraries or DNA; and PCR amplification of appropriate libraries, or RNAor DNA using oligonucleotide primers of the present invention (genomicand/or cDNA libraries can be used). To isolate flea saliva proteinnucleic acid molecules, preferred cDNA libraries include cDNA librariesmade from unfed whole flea, fed whole flea, fed flea midgut, unfed fleamidgut, and flea salivary gland. Techniques to clone and amplify genesare disclosed, for example, in Sambrook et al., ibid. The Examplessection includes examples of the isolation of cDNA sequences encodingflea saliva proteins of the present invention.

[0135] The present invention also includes nucleic acid molecules thatare oligonucleotides capable of hybridizing, under stringent conditions,with complementary regions of other, preferably longer, nucleic acidmolecules of the present invention that encode at least a portion of aflea saliva product, or a homologue thereof (e.g., saliva products ofother ectoparasites), contained in the saliva extracts FS-1, FS-2 orFS-3. A preferred oligonucleotide is capable of hybridizing, understringent conditions, with a nucleic acid molecule that is capable ofencoding at least a portion of one or more of the proteins fspA, fspB,fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI,fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3,or homologues thereof. As such, certain preferred oligonucleotides arecapable of hybridizing to a nucleic acid molecule capable of encoding aprotein having at least a portion of one or more of the following aminoacid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54 and/or SEQ ID NO: 56, orhomologues thereof. Certain preferred oligonucleotides are capable ofhybridizing to nucleic acid molecules including nucleic acid sequencesrepresented by SEQ ID NO: 20, SEQ ID: 24, SEQ ID NO: 32, SEQ ID NO: 34,SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 55 or complements thereof.

[0136] Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimal size of such oligonucleotides is thesize required to form a stable hybrid between a given oligonucleotideand the complementary sequence on another nucleic acid molecule of thepresent invention. Minimal size characteristics are disclosed herein.The size of the oligonucleotide must also be sufficient for the use ofthe oligonucleotide in accordance with the present invention.Oligonucleotides of the present invention can be used in a variety ofapplications including, but not limited to, as probes to identifyadditional nucleic acid molecules, as primers to amplify or extendnucleic acid molecules or in therapeutic applications to inhibit, forexample, expression of saliva proteins by ectoparasites. Suchtherapeutic applications include the use of such oligonucleotides in,for example, antisense-, triplex formation-, ribozyme- and/or RNAdrug-based technologies. The present invention, therefore, includes sucholigonucleotides and methods to interfere with the production ofectoparasite saliva proteins by use of one or more of such technologies.

[0137] The present invention also includes a recombinant vector, whichincludes an ectoparasite saliva protein nucleic acid molecule of thepresent invention inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to ectoparasite saliva protein nucleicacid molecules of the present invention. The vector can be either RNA orDNA, either prokaryotic or eukaryotic, and typically is a virus or aplasmid. Recombinant vectors can be used in the cloning, sequencing,and/or otherwise manipulating of ectoparasite saliva protein nucleicacid molecules of the present invention. One type of recombinant vector,herein referred to as a recombinant molecule and described in moredetail below, can be used in the expression of nucleic acid molecules ofthe present invention. Preferred recombinant vectors are capable ofreplicating in the transformed cell.

[0138] A preferred nucleic acid molecule to include in a recombinantvector of the present invention is a nucleic acid molecule that encodesat least a portion of at least one flea saliva product, or a homologuethereof (e.g., saliva products of other ectoparasites), contained in thesaliva extracts FS-1, FS-2 or FS-3. A particularly preferred nucleicacid molecule to include in a recombinant vector is capable of encodingat least a portion of one or more of the proteins fspA, fspB, fspC1,fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1,fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3, orhomologues thereof. As such, also included are nucleic acid moleculesthat encode a protein having at least a portion of one or more of thefollowing amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54 and/or SEQ ID NO:56, or homologues thereof, and nucleic acid molecules including at leasta portion of a nucleic acid sequence represented by SEQ ID NO: 20, SEQID NO: 24, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 50, SEQ ID NO: 52and/or SEQ ID NO: 55.

[0139] In one embodiment, an isolated ectoparasite saliva protein of thepresent invention is produced by culturing a cell capable of expressingthe protein under conditions effective to produce the protein, andrecovering the protein. A preferred cell to culture is a recombinantcell that is capable of expressing the ectoparasite saliva protein, therecombinant cell being produced by transforming a host cell with one ormore nucleic acid molecules of the present invention. Transformation ofa nucleic acid molecule into a cell can be accomplished by any method bywhich a nucleic acid molecule can be inserted into the cell.Transformation techniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a host cell include nucleic acid molecules that encode atleast a portion of a flea saliva product, or a homologue thereof (e.g.,saliva products of other ectoparasites), contained in the salivaextracts FS-1, FS-2 or FS-3. Particularly preferred nucleic acidmolecules with which to transform a host cell are as disclosed hereinfor including in recombinant vectors of the present invention.

[0140] Suitable host cells to transform include any cell that can betransformed and that can express the introduced ectoparasite salivaprotein. Such cells are, therefore, capable of producing ectoparasitesaliva proteins of the present invention after being transformed with atleast one nucleic acid molecule of the present invention. Host cells canbe either untransformed cells or cells that are already transformed withat least one nucleic acid molecule. Suitable host cells of the presentinvention can include bacterial, fungal (including yeast), insect,animal and plant cells. Preferred host cells include bacterial, yeast,insect and mammalian cells, with bacterial (e.g., E. coli) and insect(e.g., Spodoptera) cells being particularly preferred.

[0141] A recombinant cell is preferably produced by transforming a hostcell with one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of aspecified nucleic acid molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal, insect,animal, and/or plant cells. As such, nucleic acid molecules of thepresent invention can be operatively linked to expression vectorscontaining regulatory sequences such as promoters, operators,repressors, enhancers, termination sequences, origins of replication,and other regulatory sequences that are compatible with the recombinantcell and that control the expression of nucleic acid molecules of thepresent invention. As used herein, a transcription control sequenceincludes a sequence which is capable of controlling the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressor equences.Suitable transcription control sequences include any transcriptioncontrol sequence that can function in at least one of the recombinantcells of the present invention. A variety of such transcription controlsequences are known to those skilled in the art. Preferred transcriptioncontrol sequences include those which function in bacterial, yeast,helminth, insect and mammalian cells, such as, but not limited to, tac,lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (λ) (such asλp_(L) and λp_(R) and fusions that include such promoters),bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6,bacteriophage SP01, metallothionein, alpha mating factor, Pichia alcoholoxidase, alphavirus subgenomic promoters (such as Sindbis virussubgenomic promoters), baculovirus, Heliothis zea insect virus, vacciniavirus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirusactin, retroviral long terminal repeat, Rous sarcoma virus, heat shock,phosphate and nitrate transcription control sequences as well as othersequences capable of controlling gene expression in prokaryotic oreukaryotic cells. Additional suitable transcription control sequencesinclude tissue-specific promoters and enhancers as well aslymphokine-inducible promoters (e.g., promoters inducible by interferonsor interleukins). Transcription control sequences of the presentinvention can also include naturally occurring transcription controlsequences naturally associated with a DNA sequence encoding anectoparasite saliva protein.

[0142] Expression vectors of the present invention may also containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed ectoparasite saliva protein to be secreted from thecell that produces the protein. Suitable signal segments include anectoparasite saliva protein signal segment or any heterologous signalsegment capable of directing the secretion of an ectoparasite salivaprotein, including fusion proteins, of the present invention. Preferredsignal segments include, but are not limited to, tissue plasminogenactivator (t-PA), interferon, interleukin, growth hormone,histocompatibility and viral envelope glycoprotein signal segments.

[0143] Expression vectors of the present invention may also containfusion sequences which lead to the expression of inserted nucleic acidmolecules of the present invention as fusion proteins. Inclusion of afusion sequence as part of an ectoparasite nucleic acid molecule of thepresent invention can enhance the stability during production, storageand/or use of the protein encoded by the nucleic acid molecule.Furthermore, a fusion segment can function as a tool to simplifypurification of an ectoparasite saliva protein, such as to enablepurification of the resultant fusion protein using affinitychromatography. A suitable fusion segment can be a domain of any sizethat has the desired function (e.g., increased stability and/orpurification tool). It is within the scope of the present invention touse one or more fusion segments. Fusion segments can be joined to aminoand/or carboxyl termini of an ectoparasite saliva protein. Linkagesbetween fusion segments and ectoparasite saliva proteins can beconstructed to be susceptible to cleavage to enable straight-forwardrecovery of the ectoparasite saliva proteins. Fusion proteins arepreferably produced by culturing a recombinant cell transformed with afusion nucleic acid sequence that encodes a protein including the fusionsegment attached to either the carboxyl and/or amino terminal end of anectoparasite saliva protein.

[0144] A recombinant molecule of the present invention is a moleculethat can include at least one of any nucleic acid molecule heretoforedescribed operatively linked to at least one of any transcriptioncontrol sequence capable of effectively regulating expression of thenucleic acid molecule(s) in the cell to be transformed. A preferredrecombinant molecule includes one or more nucleic acid molecules thatencode at least a portion of a flea saliva product, or a homologuethereof (e.g., saliva products of other ectoparasites), contained in thesaliva extracts FS-1, FS-2, or FS-3. Particularly preferred nucleic acidmolecules to include in a recombinant molecule are as disclosed hereinfor including in a recombinant vector of the present invention.

[0145] A recombinant cell of the present invention includes any cellstransformed with at least one of any nucleic acid molecules of thepresent invention. A preferred recombinant cell is a cell transformedwith at least one nucleic acid molecule that encodes at least a portionof a flea saliva product, or a homologue thereof (e.g., saliva productsof other ectoparasites), contained in the saliva extracts FS-1, FS-2and/or FS-3. A preferred recombinant cell is transformed with at leastone nucleic acid molecule that is capable of encoding at least a portionof one or more of the proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2,fspE, fspF, fspG1 fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1,fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3, or homologues thereof. Assuch, also included are nucleic acid molecules that encode a proteinhaving at least a portion of one or more of the following amino acidsequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:51, SEQ ID NO: 53, SEQ ID NO: 54 and/or SEQ ID NO: 56, or homologuesthereof, and nucleic acid molecules including at least a portion of anucleic acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 24, SEQID NO: 32, SEQ ID NO: 34, SEQ ID NO: 50, SEQ ID NO: 52 and/or SEQ ID NO:55. Particularly preferred recombinant cells include E. coli transformedwith at least one of the aforementioned nucleic acid molecules.

[0146] It may be appreciated by one skilled in the art that use ofrecombinant DNA technologies can improve expression of transformednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within a host cell, the efficiencywith which those nucleic acid molecules are transcribed, the efficiencywith which the resultant transcripts are translated, and the efficiencyof post-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant protein productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing the resultant protein.

[0147] In accordance with the present invention, recombinant cells canbe used to produce an ectoparasite saliva protein of the presentinvention by culturing such cells under conditions effective to producesuch a protein, and recovering the protein. Effective conditions toproduce a protein include, but are not limited to, appropriate media,bioreactor, temperature, pH and oxygen conditions that permit proteinproduction. An appropriate, or effective, medium refers to any medium inwhich a cell of the present invention, when cultured, is capable ofproducing an ectoparasite saliva protein. Such a medium is typically anaqueous medium comprising assimilable carbohydrate, nitrogen andphosphate sources, as well as appropriate salts, minerals, metals andother nutrients, such as vitamins. The medium may comprise complexnutrients or may be a defined minimal medium.

[0148] Cells of the present invention can be cultured in conventionalfermentation bioreactors, which include, but are not limited to, batch,fed-batch, cell recycle, and continuous fermentors. Culturing can alsobe conducted in shake flasks, test tubes, microtiter dishes, and petriplates. Culturing is carried out at a temperature, pH and oxygen contentappropriate for the recombinant cell. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art.

[0149] Depending on the vector and host system used for production,resultant ectoparasite saliva proteins may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane. The phrase “recovering the protein” refers simply tocollecting the whole fermentation medium containing the protein and neednot imply additional steps of separation or purification. Ectoparasitesaliva proteins of the present invention can be purified using a varietyof standard protein purification techniques, such as, but not limitedto, affinity chromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, chromatofocusing anddifferential solubilization.

[0150] Ectoparasite saliva proteins are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. For example, an animal beingadministered dosages of ectoparasite saliva protein isolated from arecombinant cell of the present invention should exhibit no substantialtoxicity from contaminants mixed with the protein.

[0151] Ectoparasite saliva products substantially free of contaminatingmaterial can be isolated using a saliva collection apparatus of thepresent invention. A saliva collection apparatus of the presentinvention is designed to stimulate (i.e., cause) ectoparasites retainedin the container to feed, and thereby to release saliva, which iscollected separate from contaminating material.

[0152] Ectoparasites attach and feed from warm-blooded host animals. Ahost animal, as used herein, refers to an animal that ectoparasites canfeed from or on. Without being bound by theory, it is believed thatectoparasites, such as fleas, possess heat receptors which enables theectoparasite to sense a temperature differential between the warm skinof the host and the ambient air. The temperature differential stimulates(i.e., causes) the ectoparasite to feed from the warm surface (i.e.,from the warm animal skin). It is also believed that motion, vibrationand darkness can be sensed by ectoparasites, thereby encouraging them tofeed. An ectoparasite feeds by penetrating the dermis of an animal withits mouthparts, the mouthparts remaining in that position while theectoparasite secretes saliva to enhance feeding. During feeding, anectoparasite can release contaminants such as blood proteins and fecalmaterial.

[0153] A saliva collection apparatus of the present invention includes achamber and housing such that a temperature differential between thechamber and housing of the apparatus is maintained which causes orpromotes ectoparasites retained in the housing to attempt to feed fromthe chamber. When ectoparasites housed in such an apparatus attempt tofeed in accordance with the present invention, the arthropods releasesaliva which is collected in such a manner that proteins, and otherproducts, in the saliva are isolated substantially free of contaminatingmaterial. In order to collect saliva substantially free of contaminatingmaterial, an apparatus of the present invention also includes acollection means to capture saliva on a surface separate from thesurface which captures the contaminating material.

[0154] A saliva collection apparatus of the present invention can beused to collect saliva from any ectoparasite such as those disclosedherein. Ectoparasites of the present invention can feed on any animalsusceptible to ectoparasite infestation (i.e., a host animal), includingbut not limited to, a wide variety of vertebrates. Preferred hostanimals include mammals and birds. More preferred host animals includecats, dogs, humans, horses, rabbits, sheep, cattle, swine, goats,raccoons, ferrets, rats and opossums as well as other pets, economicfood animals and animals that are hosts for fleas that infest pets andeconomic food animals. Particularly preferred host animals are cats anddogs.

[0155] Particularly preferred ectoparasites of the present inventionfrom which to collect saliva include any suitable species of flea.Preferred fleas include fleas capable of infesting cats and dogs. Newlyhatched fleas (i.e., recently emerged from a pupal state) that have nothad a first blood meal are preferred for the following reasons: Becausenewly emerged fleas have not had a first blood meal, such fleas attemptto feed. Since newly emerged fleas have not had a blood meal, they alsodo not release as much contaminating material as do fed fleas. Newlyemerged fleas live longer without a blood meal than do fleas which havehad at least one blood meal. It should be noted that fed fleas can alsobe used with an apparatus of the present invention.

[0156] It will be obvious to one of skill in the art that a salivacollection apparatus of the present invention is useful for collectingsaliva from any ectoparasite. For the purpose of illustration, a fleasaliva collection apparatus of the present invention is described indetail below. Such description is not intended, in any way, to limit thescope of the present invention. It is within the skill of one in the artto collect saliva from other ectoparasites in a straightforward mannerbased on methods to collect saliva from fleas.

[0157] One embodiment of the present invention is a saliva collectionapparatus that includes a chamber and a housing operatively connected toan interface in such a manner that a temperature differential ismaintained between the chamber and the housing. The interface includes acollection means and a barrier means positioned such that, in order toattempt to feed, flea mouthparts penetrate the barrier means prior tothe collection means. The temperature differential between the chamberand the housing is a difference in temperature suitable to attract fleasretained in the housing to attempt to feed through the interface and,thereby deposit saliva products on the collection means. Due to therelative positioning of the collection means and the barrier means,contaminating material is deposited on the barrier means.

[0158] A flea saliva collection apparatus of the present inventionincludes a housing. A housing can comprise any material capable ofretaining fleas that provides structural support and that can beconnected to a retaining means. The housing is preferably made of amaterial capable of withstanding cleaning and/or sterilizationprocedures commonly used by those skilled in the art. As such, thehousing can be reused. Preferred housing materials of the presentinvention include, but are not limited to, plastic, metal, rubber, woodand glass materials and combinations of such materials. More preferredhousing materials include plastic and metal materials with plasticmaterials being even more preferred. Preferred plastic materials includeplexiglass, teflon, nylon and polycarbonate. A particularly preferredplastic material is plexiglass, or other durable, break-resistantplastic, preferably clear so as to allow viewing of fleas inside thecontainer.

[0159] In accordance with the present invention, the size of a housingof the present invention is such that the housing can support a desirednumber of fleas without overcrowding. Both surface area and the volumeof the housing can be important. The size of the housing can varyaccording to the number of fleas to be retained in the housing.Preferably, the size of the housing is sufficient to maintain from about1,000 fleas to about 6,000 fleas per housing for about 72 hours, morepreferably from about 2,000 fleas to about 5,000 fleas per housing forabout 72 hours, and even more preferably from about 3,000 fleas to about4,000 fleas per housing for about 72 hours.

[0160] A suitable height for a housing of the present invention is aheight that is sufficiently high to allow room for fleas to move aboutwhile feeding. The height of a housing for fleas is preferably fromabout 1.0 centimeters (cm) to about 3.0 cm, more preferably from about1.5 cm to about 2.5 cm, and even more preferably from about 1.8 cm toabout 2.2 cm.

[0161] The shape of a housing of the present invention can be any shapehaving at least one flat surface suitable for feeding by fleas containedwithin the housing. A housing of the present invention is preferablyshaped as a cylinder, a box having four or more sides, a half-dome, or ahalf cylinder. A particularly preferred shape is a short cylinder.

[0162] The diameter of a preferred housing of the present invention canvary widely. Different diameter containers can be used according to, forexample, the number of fleas to be placed into the housing withoutovercrowding. The interior diameter of a rounded housing of the presentinvention is preferably from about 4.0 cm to about 5.5 cm, morepreferably from about 4.5 cm to about 5.5 cm, and even more preferablyabout 5.0 cm.

[0163] According to the present invention, the size, shape, height, anddiameter of the housing can vary for different ectoparasites dependingupon the size and number of arthropods retained in the housing.

[0164] In accordance with the present invention, a housing isoperatively connected to a retaining means and an exchange means. Asused herein, “operatively connected” refers to combining portions of asaliva collection apparatus of the present invention in such a mannerthat fleas can be retained within the apparatus and can deposit salivaon the collection means. A retaining means of the present invention ispenetrable by the mouthparts of fleas. A retaining means of the presentinvention can comprise any material or combination of materials that issuitable for retaining fleas and through which fleas can feed (i.e., theretaining means is penetrable by flea mouthparts). As such, theretaining means may comprise a material having openings sufficientlylarge (i.e., large enough) for flea mouthparts to penetrate, butsufficiently small (i.e., small enough) so as to effectively preventloss of any fleas retained therein. Preferred retaining means comprise amaterial having openings of from about 0.25 millimeters (mm) to about0.50 mm, more preferably having openings of from about 0.30 mm to about0.50 mm, and even more preferably having openings of from about 0.35 mmto about 0.45 mm. One of skill in the art will recognize that the sizeof the openings can vary according to the type of ectoparasite retainedin the housing of an apparatus. For example, maintenance of particularlysmall ectoparasites such as, but not limited to, lice may requireretaining means having smaller openings. conversely, a retaining meansfor hard ticks, which are ectoparasites that cement their mouthpartsinto the host animal, require larger openings, preferably in the rangeof about 1 mm.

[0165] Preferred materials for use as retaining means include, but arenot limited to, metallic mesh, nylon mesh, plastic film, cloth andcombinations of such materials. More preferred retaining means includenylon mesh and metal mesh, and an even more preferred retaining meansincludes nylon mesh. The collection apparatus can be retrofitted with avariety of retaining means. Preferred retaining means are reusable.

[0166] An exchange means of the present invention can comprise anymaterial or combination of materials capable of maintaining a permissiveenvironment for fleas within the housing by allowing the exchange ofgas, humidity and heat between the interior environment of the housingand the environment exterior to the housing. The housing can beretrofitted with different exchange means having different gas, humidityand heat permeabilities. As used herein, the term gas refers to anyatmospheric gases required for flea survival, including, but not limitedto, carbon dioxide, oxygen, and nitrogen. Gas can also refer to gaseousproducts produced by fleas while maintained in a housing of the presentinvention, such as gaseous products of metabolism including expirationsor gases from feces.

[0167] Exchange means of the present invention are comprised ofmaterials having openings that are sufficiently large to allow gas, heatand humidity to escape, but sufficiently small so as to effectivelyprevent loss of fleas. Preferred exchange means comprise a materialhaving openings of from about 0.10 millimeters (mm) to about 0.45 mm,more preferably having openings of from about 0.10 mm to about 0.30 mm,and even more preferably having openings of from about 0.13 mm to about0.15 mm.

[0168] Preferred materials to use as an exchange means include, but arenot limited to, metallic mesh, nylon mesh, plastic, cloth andcombinations of such materials. More preferred exchange means includenylon mesh, metal mesh, and combinations of such materials and an evenmore preferred exchange means includes nylon mesh. Preferred exchangematerials are reusable.

[0169] In accordance with the present invention, an apparatus includes achamber operatively connected to a housing. A chamber of the presentinvention is capable of maintaining an internal temperature suitable tocreate a temperature differential between a housing and a chamber of anapparatus which promotes deposition of saliva by fleas retained in thehousing on a collection means of an apparatus. A preferred chamber isalso capable of maintaining an internal humidity level suitable for thesurvival of ectoparasites contained in the apparatus (e.g., suitable toprevent desiccation of the ectoparasites). A chamber of the presentinvention is also capable of being attached to an artificial feedingsystem as described in detail in the Examples. A chamber can compriseany material capable of maintaining suitable temperature and humiditylevels within the chamber. A chamber is preferably made of a materialcapable of withstanding cleaning or sterilization procedures commonlyused by those skilled in the art. As such, a chamber can be reused.Preferred chamber materials of the present invention include, but arenot limited to, glass, plastic, metal, rubber, and wood materials andcombinations of such materials. More preferred chamber materials includeglass and plastic materials with glass materials being even morepreferred.

[0170] In accordance with the present invention, the size of a chamberof the present invention is such that the chamber can maintain asuitable temperature level to stimulate fleas to deposit saliva on thecollection means of the apparatus. The size of the chamber can varyaccording to the amount of blotting material (as described in detailbelow) to be placed in the chamber, the diameter of the collection meansto be attached to the chamber or whether the chamber is to be attachedto an artificial feeding system as described in detail in the Examples.Preferably, the height of a chamber of the present invention is highenough to allow a suitable amount of blotting material to be placed inthe chamber, such that the blotting material maintains a humidity levelin the chamber suitable for flea survival. The height of a chamber ispreferably from about 1.0 cm to about 7.0 cm, more preferably from about2.0 cm to about 6.0 cm, and even more preferably from about 3.0 cm toabout 5.0 cm.

[0171] In accordance with the present invention, the shape of a chambercan be any shape having at least one open end to which an interface ofthe present invention can be attached. A chamber of the presentinvention is preferably shaped as a cylinder open at both ends or acylinder open at one end. A particularly preferred shape is a cylinderopen at both ends.

[0172] The diameter of a preferred chamber of the present invention canvary widely. Different diameter chambers can be used according to, forexample, the diameter of the interface to be attached to the chamber orthe diameter of the housing to be attached to the chamber. The interiordiameter of a chamber of the present invention is preferably from about2.0 cm to about 6.5 cm, more preferably from about 3.0 cm to about 5.5cm, and even more preferably from about 4.0 cm to about 4.5 cm.

[0173] A chamber of the present invention can contain a blotting meanssuitable for maintaining a humidity level in the chamber suitable forflea survival. Methods for maintaining suitable humidity levels aredescribed in detail below. A chamber of the present invention cancontain food or water, but preferably is humid (i.e., damp but not wet)and does not contain food.

[0174] A saliva collection apparatus of the present invention includesan interface. An interface of the present invention includes meanscapable of collecting saliva products substantially free ofcontaminating material. As such, an interface of the present inventionis penetrable by flea mouthparts but capable of keeping contaminatingmaterial, such as blood and fecal material, separate from flea salivaproducts secreted by fleas as they attempt to feed. An interface of thepresent invention comprises a means for collecting saliva products and ameans for creating a barrier between contaminating material andcollected saliva products.

[0175] A collection means of the present invention can be of anymaterial capable of collecting (i.e., adsorbing) at least a portion ofsaliva proteins deposited (i.e., secreted) by retained fleas that areattempting to feed through the interface. In addition, a collectionmeans of the present invention is capable of collecting salivacomponents other than saliva proteins deposited by fleas attempting tofeed through the interface. The collection means is such that salivaproducts not only can bind to the collection means but also can beeluted (i.e., extracted) therefrom upon exposure to a suitable eluent(i.e., extractant). As such, preferred collection means materials of thepresent invention include materials that are hydrophobic and have a lowbinding capacity since saliva components are easily eluted from suchmaterial. The material of a collection means of the present inventionshould also be capable of being penetrated by the mouthparts of fleas.

[0176] Preferred collection means materials of the present inventioninclude, but are not limited to, nylon, nitrocellulose, CM-derivatized,diethylaminoethyl (DEAE)-derivatized, paper, polysulfone, celluloseester, polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF)membranes. A particularly preferred anion exchange membrane collectionmeans material includes DE-81 chromatography paper, which can beobtained from Whatman, Inc., Clifton, N.J. Particularly preferredcollection means materials include PVDF. A preferred PVDF collectionmeans material includes Durapore™.

[0177] The shape of a collection means of the present invention can varyaccording to the shape of the chamber to which the collection means isto be attached. A preferred shape of a collection means includes, but isnot limited to, a round shape or a box-like shape having four or moresides, with a round shape being more preferred.

[0178] The size of a collection means of the present invention can alsovary according to the size of the chamber to which the collection meansis to be attached. The size of a collection means is preferably largerthan the open end of a chamber, thereby preventing the collection meansfrom passing into the chamber. The size of a collection means ispreferably from about 2.2 cm to about 6.5 cm in diameter, morepreferably from about 3.2 cm to about 5.7 cm, and even more preferablyfrom about 4.2 cm to about 4.7 cm.

[0179] A saliva collection apparatus of the present invention providesfor a novel barrier means which enables collection of ectoparasitesaliva substantially free of contaminating material. A barrier means ofthe present invention can be any material capable of substantiallypreventing contaminating material from contacting the collection means(i.e., substantially prevents the passage of contaminating material suchas flea fecal material and blood products through the collection means),but is also capable of being penetrated by the mouthparts of fleas andof allowing the passage of saliva through the barrier means. Preferably,the thickness of a barrier means material of the present invention ismicrons thick. Preferred barrier means materials of the presentinvention include, but are not limited to, very thin plastic, teflon,cloth, paper, paraffin and wax materials. More preferred barrier meansmaterials of the present invention include stretched plastic, with SaranWrap™ and particularly Parafilm™, stretched very thin (i.e., as thin ascan be stretched by machine and/or hand), being even more preferred.

[0180] The size of a barrier means of the present invention can varyaccording to the size of the chamber to which the barrier means is to beattached. The size of the barrier means preferably is sufficiently largethat the barrier means can extend up the sides of a chamber of thepresent invention, thereby enabling the barrier means to be secured tothe chamber. The size of the barrier means is sufficiently small suchthat the barrier means does not interfere with, for example, the abilityof the saliva collection apparatus containing the chamber to be attachedto an artificial feeding system.

[0181] According to the present invention, a collection means and abarrier means are operatively connected to a chamber of a salivacollection apparatus in such a manner that fleas retained in the housingof such apparatus are capable of penetrating both the barrier means andthe collection means to deposit saliva on the collection means. Acollection means of the present invention preferably is removablyattached to a site on a chamber by a barrier means. A preferred site ofattachment of a collection means and a barrier means is the portion of achamber designed to interface with a housing. A more preferred site ofattachment of a collection means and a barrier means is the open end ofa chamber.

[0182] A saliva collection apparatus of the present invention can alsoinclude a blotting means. A blotting means of the present invention iscapable of maintaining a humidity within the apparatus suitable for fleasurvival and, as such, is capable of retaining liquid for the period oftime fleas are retained in the apparatus. As such, a blotting means canbe any suitable absorbent material. Preferred blotting means includenatural and synthetic sponges, foam, paper, cloth, and agarose. Morepreferred blotting means material include sponges and paper, with filterpaper being even more preferred. In a particularly preferred embodiment,one or more pieces of VWR Blotting Pads #320 (available from VWRScientific, Denver, Colo.) comprise a blotting means.

[0183] As stated above, a saliva collection apparatus of the presentinvention is capable of maintaining a temperature differential between ahousing and a chamber of the apparatus. A suitable temperaturedifferential within an apparatus of the present invention includes atemperature differential which stimulates fleas retained in theapparatus to penetrate the interface of the apparatus and deposit salivaon the collecting means. Preferred temperatures in a chamber of thepresent invention range from about 20° C. to about 45° C., whereaspreferred temperatures in a housing of the present invention range fromabout 5° C. to about 35° C. In a preferred embodiment, the temperaturein the chamber ranges from about 35° C. to about 40° C. and thetemperature in the housing ranges from about 10° C. to about 30° C. Aparticularly preferred chamber temperature ranges from about 35° C. toabout 37° C.; and a particularly preferred housing chamber temperatureis from about 20° C. to about 27° C.

[0184] The survival of ectoparasites can be affected by humidity. Assuch, the humidity level in a housing of an apparatus of the presentinvention is suitable for maintaining the survival of ectoparasitesretained therein. Suitable relative humidity levels within an apparatusof the present invention can vary depending upon the ectoparasitecontained within the apparatus. As used herein, relative humidity refersto the degree of atmospheric water vapor relative to the maximum degreeof atmospheric water vapor that results in precipitation. Thus, relativehumidity is expressed in percent humidity, wherein 100% humidityrepresents saturation of atmospheric water vapor. Preferred humiditylevels in a chamber of the present invention range from about 50% toabout 100%, whereas preferred humidity levels in a housing of thepresent invention range from about 40% to about 60%. In a preferredembodiment, the humidity levels in the chamber ranges from about 50% toabout 94% and the humidity level in the housing is about 50%.

[0185] Another embodiment of the present invention is the use ofcontrasting colors to attract fleas. For example, at least one surfaceof a collection apparatus of the present invention can be of a colorsufficiently dark to attract fleas to penetrate the interface of theapparatus. Without being bound by theory, it is believed that fleas arecapable of sensing light from dark and preferably tend to feed towards adark surface. Therefore, according to the present invention, a chambercan be darker than a housing, thereby attracting fleas to the interfacebetween the chamber and the housing. Suitable dark colors include colorsranging from black to light brown, preferably black.

[0186] A preferred embodiment of a collection apparatus of the presentinvention is depicted in FIGS. 4A and 4B. A saliva collection apparatus(2) is separable into a housing (4) and a chamber (6). A cross-sectionof a saliva collection apparatus of the present invention (2) is shownin FIG. 4A. Referring to FIGS. 4A and 4B, the housing (4) has anopen-ended cylinder having a first portion (8) and a second portion(10). The second portion (10) of the housing (4) has an outer diameter(12) and an inner diameter (14). An exchange means (16) is operativelyattached to one end of the first portion (8) of the housing (4) and aretaining means (18) is attached at the opposite end of the firstportion (8), between the first portion (8), and the outer diameter (12)and inner diameter (14) of the second portion (10). The exchange means(16) and retaining means (18) are attached in a manner that preventsfleas from escaping. Means of attaching an exchange means (16) and aretaining means (18) to the first portion (8) of the housing (4)include, but are not limited to, rubber cement, glue, tape, solder andaraldite. A preferred means of attachment is rubber cement.

[0187] The chamber (6) of the saliva collection apparatus (2) has anopen ended cylinder having a top end (20) and a bottom end (22). The topend (20) has a suitable diameter to enable the attachment of the chamber(6) to an artificial feeding system such as that described in detail inthe Examples. The bottom end (22) has a suitable diameter such that thebottom end (22) can be reversibly attached to the housing (4) in such amanner as to provide a sliding fit. The bottom end (22) is covered by acollection means (24). The collection means (24) has a larger diameterthan the inner circumference of the bottom end (22) of the chamber (6),thereby preventing the collection means (24) from passing into the innerspace (26) of the chamber (6). Preferably, the diameter of thecollection means (24) is not greater than the outer circumference of thebottom end (22) of the chamber (6). The collection means (24) can beconnected to the bottom end (22) in order to provide a detachableconnection, thereby facilitating removal of the collection means (24)from the saliva collecting apparatus (2) to recover saliva products fromthe collection means (24).

[0188] A collection means (24) attached to the bottom end (22) of achamber (6) is covered by a barrier means (28). The barrier means (28)is operatively connected to a chamber (6) in such a manner that a sealis formed preventing contact of the collection means (24) bycontaminating material deposited by fleas. The barrier means (28) can beconnected to the chamber (6) in order to provide a detachableconnection. Preferably, the barrier means (28) comprises a stretchableplastic material, such as Parafilm™, which is stretched as thin aspossible across a collection means (24) contacting the bottom end (22)of a chamber (6) and further stretched along the sidewall (30) of thechamber (6) towards the top end (20) of the chamber (6). The barriermeans (28) can be further secured to the sidewall (30) of the chamber(6) using a rubber seal (32). The rubber seal (32) detachably connectsthe portion of the barrier means (28) which meets the sidewall (30) ofthe chamber (6), thereby further securing the collection means (24) tothe chamber (6) and seal in the chamber (6) environment.

[0189] Blotting (absorbent) material can be placed in the inner space(26) at the bottom end (22) of a chamber (6) to form a blotting means(34). The blotting means (34) can comprise one or more individualblotting pads (e.g., pieces of blotting material). Preferably, theblotting means (34) is from about 2.0 mm thick to about 15.0 mm thick(when dry) when placed in a 47 cm high chamber (6), more preferably isfrom about 2.2 mm thick to about 12.5 mm thick (when dry) when placed ina 47 mm high chamber (6), and even more preferably is from about 2.45 mmthick to about 10.0 mm thick (when dry) when placed in a 47 mm highchamber (6). In a particularly preferred embodiment, the blotting means(34) comprises from about 2 to 6 pieces of VWR Blotting Pads #320, andpreferably from about 3 to 5 pieces of VWR Blotting Pads #320. Thediameter of the blotting means (34) is selected to contact the innersidewall (36) of the chamber (6). The blotting means (34) is preferablysufficiently pre-wetted to provide humidity to the chamber (6) but notso wet that liquid drips from the blotting means (34). The blottingmeans (34) is juxtaposed to the side of the collection means (24) facingthe top end (20) of the chamber (6). The blotting means (34) candirectly contact the collection means (24) in a detachable manner.

[0190] The chamber (6) is reversibly separable from the housing (4). Thechamber (6) can be interconnected to the housing (4) in any reversiblysecure manner such as sliding, snapping or screwing together.Preferably, the chamber (6) slides into the housing (4) and is securedusing rubber bands.

[0191] The relative height dimensions of the chamber (6) can varyrelative to the housing (4). Typically, the height dimension of thechamber (6) is greater than the housing (4). Preferably, the height ofthe chamber (6) ranges from about 1.0 cm to about 7.0 cm, morepreferably from about 2.0 cm to about 6.0 cm, and even more preferablythe from about 3.0 cm to about 5.0 cm. The height of the housing (4)preferably ranges from about 1.0 cm to about 3.0 cm, more preferablyfrom about 1.5 cm to about 2.5 cm, and even more preferably from about1.8 cm to about 2.2 cm.

[0192] One embodiment of the present invention is a method to collectsaliva products from ectoparasites using an apparatus of the presentinvention. Such a method is particularly advantageous because it enablesisolation of ectoparasite saliva, including saliva proteins,substantially free of contaminating material. As such, the method can beused, for example, to characterize ectoparasite saliva proteins and toisolate ectoparasite saliva proteins for diagnostic and therapeutic use.

[0193] One embodiment of the present method includes the steps of: (a)collecting ectoparasite saliva products on a collection means within asaliva collection apparatus which contains ectoparasites in the housingof the apparatus; and (b) extracting (i.e., eluting) the collectedectoparasite saliva products from the collection means with a solutionto form an extracted product-containing solution. Such an extractedsolution can be used directly as a formulation of the present inventionor can be submitted to further steps of fractionation and/orpurification as described in detail herein, to form additionalformulations of the present invention. Examples of such extractedsolutions include FS-1, FS-2 and FS-3.

[0194] In accordance with the present invention, a saliva collectionapparatus containing the ectoparasites has an interface between thechamber and the housing comprising a collection means capable ofcollecting at least a portion of saliva products deposited byectoparasite retained in the apparatus and a barrier means capable ofsubstantially preventing contaminating material from contacting thecollection means. The ectoparasites contained in the apparatus aremaintained under conditions such that there is a temperaturedifferential between the chamber and the housing; that is, the chamberof the apparatus has a temperature warmer than the temperature of thehousing containing the ectoparasite, such that the warmer temperature inthe chamber attracts the ectoparasites retained in the housing toattempt to penetrate the barrier means and collection means, therebydepositing saliva products on the collection means.

[0195] In one embodiment, the method of collecting saliva productsincludes pre-wetting a collection means of the present invention priorto positioning the collection means in an apparatus of the presentinvention. A pre-wetting solution suitable for the present invention iscapable of facilitating the adsorption (i.e., collection) of salivaproducts in such a manner that the products can also be extracted duringan extraction step (i.e., when exposed to an appropriate solvent). Asuitable pre-wetting solution of the present invention includes anybuffer that is non-toxic to ectoparasites and has a physiological pH.Examples of suitable buffers include, phosphate buffered saline, water,phosphate buffer, HEPES buffer(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffered saline),TES buffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer(Tris-acetate-EDTA). A preferred prewetting solution includes sterilewater containing 50 U/ml penicillin and 50 μg/ml streptomycin.

[0196] When an apparatus used to collect saliva products includes ablotting means, that blotting means should be moistened either prior toor following placement of the blotting means into the chamber. Preferredmoistening solutions include, but are not limited to, water, phosphatebuffered saline, phosphate buffer, tris buffer, HEPES buffer, TEA bufferand TES buffer. More preferred moistening solutions include water and 50U/ml penicillin and 50 μg/ml streptomycin. According to the presentinvention, a blotting means is sufficiently moistened to producehumidity in a chamber but not to drip liquid from which ectoparasitesretained in the apparatus can drink. In a preferred embodiment, ablotting means which is about 4.0 cm in diameter and about 2.5 mm thickis moistened with about 2.3 milliliters (ml) of moistening solution.

[0197] In one embodiment, a pre-determined number of ectoparasites areintroduced into the housing of an apparatus of the present invention.The number of ectoparasites to be introduced into a housing can varywith the size of the housing and should be a number that will not hinderthe ability of an ectopaiasite to deposit saliva on a collection meansof the present invention.

[0198] In a preferred embodiment of the present invention, fleas areadded to an apparatus of the present invention. Suitable and preferrednumbers of fleas to introduce into a housing are heretofore disclosed.In particular, fleas newly emerged from the pupal stage are used. Suchfleas can be raised as described in Wade et al., pp 186-190, 1988, J.MedEntomol., vol 25. Preferably, such fleas have not had a blood meal.Fleas can be loaded into an apparatus by placing the fleas in anaquarium and aspirating them into the housing under vacuum. Additionaloptional components suitable for the maintenance of fleas can be addedto the container, such as animal hair, and dry tissue.

[0199] In a preferred embodiment, at least one apparatus of the presentinvention having fleas contained in the housing of the apparatus isattached to an artificial feeding system such as disclosed herein. Theapparatus can remain attached to the feeding system as long as fleascontinue to release saliva while penetrating a collection means.Preferably, fleas are maintained in the apparatus attached to thefeeding system from about 12 hours to about 120 hours, more preferablyfrom about 24 hours to about 96 hours, and even more preferably forabout 72 hours since fleas essentially stop secreting saliva by aboutthat time. In accordance with the method of the present invention,preferably at least about 80 micrograms (μg), more preferably at leastabout 90 μg and even more preferably at least about 200 μg, of fleasaliva protein can be collected from about 10⁶ flea-hour when measuredusing a Bio-Rad Bradford assay (available from Bio-Rad, Hercules,Calif.).

[0200] According to the present invention, ectoparasite saliva productscan be extracted using a solvent capable of extracting saliva productsfrom a collection means of the present invention, preferably in a formsuch that the functional activities of the eluted products aremaintained. If functional activity of flea saliva proteins, for exampleis not maintained, it is within the scope of the invention to re-foldproteins to regain functionality using techniques known to those ofskill in the art. Suitable extraction solvents include, but are notlimited to, phosphate buffered saline, phosphate buffered salinecontaining sodium chloride, TFA in acetonitrile, chaotropic agents,detergents, organics, salts or combinations thereof. Preferredextraction solvents include phosphate buffered saline, phosphatebuffered saline containing sodium chloride, acetonitrile and TFA inacetonitrile. More preferred extraction solvents include 1 M NaCl inphosphate buffered saline, 0.1% TFA in 50% acetonitrile, 1% TFA in 50%acetonitrile, 12.8% acetonitrile and 50% acetonitrile. Suitableextraction times for eluting proteins and other products from acollection means are described in detail in the Examples.

[0201] Further purifications of saliva proteins extracted from acollection means of the present invention can be performed byfractionating the extracted product-containing solution to obtainseparated peak fractions and recovering at least one of the peakfractions substantially free of the remaining fractions to obtain aformulation of ectoparasite saliva proteins. In a preferred embodiment,proteins contained in extracted saliva products of the present inventionare further resolved by submitting the extract to HPLC purification toobtain peak fractions. In a particularly preferred embodiment, extractedsaliva proteins of the present invention are further resolved by HPLC toobtain the peak fractions shown in FIG. 2. Details regarding theextraction and resolution of such proteins are presented in theExamples.

[0202] According to the present invention, a formulation comprising atleast one ectoparasite saliva product of the present invention or amimetope thereof, can be used to identify animals that are susceptibleto or have allergic dermatitis.

[0203] In accordance with the present invention, a “mimetope” refers toany compound that is able to mimic the ability of an isolatedectoparasite saliva product of the present invention to carry out itsfunction (e.g., anti-coagulation, anti-complement, vasodialators,proteases, acid phosphatases or detecting and/or treating thehypersensitivity of an animal susceptible to or having allergicdermatitis). A mimetope can be a peptide that has been modified todecrease its susceptibility to degradation but that still retains thedesired activity. Other examples of mimetopes include, but are notlimited to, carbohydrate-based compounds, lipid-based compounds, nucleicacid-based compounds, natural organic compounds, synthetically derivedorganic compounds, anti-idiotypic antibodies and/or catalyticantibodies, or fragments thereof. Mimetopes of the present invention canalso include non-proteinaceous portions of ectoparasite saliva productshaving allergenic and/or antigenic activity (e.g., carbohydrate moietiesassociated with ectoparasite saliva proteins). A mimetope can beobtained by, for example, screening libraries of synthetic compounds forcompounds capable of altering the ability of ectoparasites to feed, orof detecting and/or treating allergic dermatitis resulting from thebites of ectoparasites. A mimetope can also be obtained by, for example,rational drug design. In a rational drug design procedure, thethree-dimensional structure of a compound of the present invention canbe analyzed by, for example, nuclear magnetic resonance (NMR) or x-raycrystallography. The three-dimensional structure can then be used topredict structures of potential mimetopes by, for example, computermodelling. The predicted mimetope structures can then be produced by,for example, chemical synthesis, recombinant DNA technology, or byisolating a mimetope from a natural source (e.g., plants, animals,bacteria and fungi).

[0204] One embodiment of the present invention is an in vivo test thatis capable of detecting whether an animal is hypersensitive toectoparasite saliva products. An in vivo test of the present inventioncan initially be used to determine if an animal is hypersensitive toectoparasite saliva products and then used to determ ne if an animal ishypersensitive to a particular ectoparasite saliva component, inparticular to an ectoparasite saliva protein. An in vivohypersensitivity test of the present invention is particularly usefulfor identifying animals susceptible to or having allergic dermatitis. Anin vivo hypersensitivity test of the present invention is even moreuseful for identifying animals susceptible to or having FAD. A suitablein vivo hypersensitivity test of the present invention can be, but isnot limited to, a skin test comprising administering (e.g.,intradermally injecting or superficial scratching) an effective amountof a formulation containing at least one ectoparasite saliva product, ora mimetope thereof. Methods to conduct skin tests of the presentinvention are known to those of skill in the art and are brieflydisclosed herein.

[0205] Suitable formulations to use in an in vivo skin test includeectoparasite saliva components (i.e., saliva products collected on, andremaining absorbed to, a collection means of the present invention,ectoparasite saliva extracts, and one or more isolated ectoparasitesaliva proteins). A preferred formulation includes extracts and one ormore isolated proteins.

[0206] A suitable amount of ectoparasite saliva product for use in askin test of the present invention can vary widely depending on theallergenicity of the product used in the test and on the site at whichthe product is delivered. Suitable amounts of ectoparasite salivaproducts for use in a skin test of the present invention include anamount capable of forming reaction, such as a detectable wheal orinduration (hardness) resulting from an allergic reaction to theproduct. Preferred amounts of ectoparasite saliva extracts or proteinsfor use in a skin test of the present invention range from about 1nanogram (ng) to about 500 micrograms (μg), more preferably from about 5ng to about 300 μg, and even more preferably from about 10 ng to about50 μg of ectoparasite saliva extracts or proteins. It is to beappreciated by those of skill in the art that such amounts will varydepending upon the allergenicity of the extracts and/or protein(s) beingadministered.

[0207] According to the present invention, ectoparasite saliva productsof the present invention can be combined with an immunopotentiator(e.g., carriers or adjuvants of the present invention as defined indetail below). A novel aspect, however, of the present invention is thatan ectoparasite saliva product of the present invention can induce ahypersensitive response in the absence of an immunopotentiator.

[0208] A skin test of the present invention further comprisesadministering a control solution to an animal. A control solution caninclude a negative control solution and/or a positive control solution.A positive control solution of the present invention contains aneffective amount of at least one compound known to induce ahypersensitive response when administered to an animal. A preferredcompound for use as positive control solution includes, but is notlimited to, histamine. A negative control solution of the presentinvention can comprise a solution that is known not to induce ahypersensitive response when administered to an animal. As such, anegative control solution can comprise a solution having compoundsessentially incapable of inducing a hypersensitive response or simply abuffer used to prepare the formulation, such as saline. An example of apreferred negative control solution is phenolated phosphate bufferedsaline (available from Greer Laboratories, Inc., Lenoir, N.C.).

[0209] Hypersensitivity of an animal to one or more formulations of thepresent invention can be evaluated by measuring reactions (e.g., whealsize, induration or hardness; using techniques known to those skilled inthe art) resulting from administration of one or more experimentalsample(s) and control sample(s) into an animal and comparing thereactions to the experimental sample(s) with reactions resulting fromadministration of one or more control solution. Preferred devices forintradermal injections include individual syringes. Preferred devicesfor scratching include devices that permit the administration of anumber of samples at one time. The hypersensitivity of an animal can beevaluated by determining if the reaction resulting from administrationof a formulation of the present invention is larger than the reactionresulting from administration of a negative control, and/or bydetermining if the reaction resulting from administration of theformulation is at least about the same size as the reaction resultingfrom administration of a positive control solution. As such, if anexperimental sample produces a reaction greater than or equal to thesize of a wheal produced by administration of a positive control sampleto an animal, then that animal is hypersensitive to the experimentalsample. Conversely, if an experimental sample produces a reactionsimilar to the reaction produced by administration of a negative controlsample to an animal, then that animal is not hypersensitive to theexperimental sample.

[0210] Preferred wheal sizes for evaluation of the hypersensitivity ofan animal range from about 16 mm to about 8 mm, more preferably fromabout 15 mm to about 9 mm, and even more preferably from about 14 mm toabout 10 mm in diameter.

[0211] Preferably, the ability or inability of an animal to exhibit animmediate hypersensitive response to a formulation of the presentinvention is determined by measuring wheal sizes from about 2 minutes toabout 30 minutes after administration of a sample, more preferably fromabout 10 minutes to about 25 minutes after administration of a sample,and even more preferably about 15 minutes after administration of asample.

[0212] Preferably, the ability or inability of an animal to exhibit adelayed hypersensitive response to a formulation of the presentinvention is determined by measuring induration and/or erythema fromabout 18 hours to about 30 hours after administration of a sample, morepreferably from about 20 hours to about 28 hours after administration ofa sample, and even more preferably at about 24 hours afteradministration of a sample. A delayed hypersensitivity response can alsobe measured using other techniques such as by determining, usingtechniques known to those of skill in the art, the extent of cellinfiltrate at the site of administration during the time periods defineddirectly above.

[0213] In a preferred embodiment, a skin test of the present inventioncomprises intradermally injecting into an animal at a given site aneffective amount of a formulation that includes flea saliva extracts(i.e., flea saliva products extracted from a collection means of thepresent invention) or at least one flea saliva protein of the presentinvention, and intradermally injecting an effective amount of a controlsolution into the same animal at a different site. It is within thescope of one of skill in the art to use devices capable of deliveringmultiple samples simultaneously at a number of sites, preferablyenabling concurrent evaluation of numerous formulations. One preferredformulation comprises flea saliva products collected in accordance withthe present invention. Also preferred are formulations comprising one ormore recombinantly produced flea saliva proteins.

[0214] Suitable flea saliva products for use with a skin test of thepresent invention include FS-1, FS-2 and/or FS-3 as well as at least aportion of at least one flea saliva product that can be isolated fromFS-1, FS-2 and/or FS-3. A preferred flea saliva product for use with askin test includes FS-1, FS-2, FS-3 and/or at least a portion of one ormore of the proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF,fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2,fspM1, fspM2, fspN1, fspN2 and fspN3, or homologues thereof. A morepreferred flea saliva product for use with a skin test includes FS-1,FS-2, FS-3 and/or at least a portion of one or more of the proteinsfspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1,fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3. A yet more preferred fleasaliva product for use with a skin test includes FS-1, FS-2, FS-3 and/orat least a portion of one or more of the proteins fspG1, fspG2, fspG3,fspH, fspM1, fspM2, fspN1, fspN2 and fspN3. Such formulations are shownin the Examples section as being able to induce FAD in dogs. A preferredpositive control sample can be a sample comprising histamine. Apreferred negative control sample can be a sample comprising diluent.

[0215] Animals suitable and preferred to test for hypersensitivity toectoparasite saliva proteins using a skin test of the present inventionare disclosed herein. Particularly preferred animals to test with a skintest of the present invention include dogs, cats and horses, with dogsand cats being even more preferred.

[0216] Another embodiment of the present invention is an in vitroimmunoabsorbent test that is capable of detecting the presence of anantibody capable of binding to one or more ectoparasite saliva productsof the present invention by contacting a putative antibody-containingsolution with a solution containing ectoparasite saliva products in sucha manner that immunocomplexes can form and be detected. Thus, an invitro immunoabsorbent test of the present invention is particularlyuseful for identifying animals susceptible to or having allergicdermatitis by demonstrating that an animal has been previously exposedto an ectoparasite saliva antigen and, therefore may be hypersensitiveto further exposure to an ectoparasite saliva antigen.

[0217] According to the present invention, an in vitro hypersensitivitytest of the present invention can be, but is not limited to, animmunoabsorbent test comprising: (a) contacting a formulation of thepresent invention with a body fluid from an animal under conditionssufficient for formation of an immunocomplex between the formulation andantibodies, if present, in the body fluid; and (b) determining theamount of immunocomplex formed, wherein formation of the immunocomplexindicates that the animal is susceptible to or has allergic dermatitis.The immunoabsorbent test is particularly useful for the detection of IgEantibodies in the body fluid, thereby indicating immediatehypersensitivity in the animal. Determining the amount of immunocomplexformed can include the step of separating depending on the mode ofdetection. Immunoabsorbent assays can be a variety of protocols and canbe set-up by those of skill in the art.

[0218] A preferred immunoabsorbent test of the present inventioncomprises a first step of coating one or more portions of a solidsubstrate with a suitable amount of one or more ectoparasite salivaproducts of the present invention or a mimetope thereof, and of coatingone or more other portions of the (or another) solid substrate with asuitable amount of positive and/or negative control solutions of thepresent invention. A preferred solid substrate of the present inventioncan include, but is not limited to, an ELISA plate, a dipstick, aradioimmunoassay plate, agarose beads, plastic beads, immunoblotmembranes and paper; a more preferred solid substrate includes an ELISAplate, a dipstick or a radioimmunoassay plate, with an ELISA plate and adipstick being even more preferred. As used herein, a dipstick refers toany solid material having a surface to which antibodies can be bound,such solid material having a stick-like shape capable if being insertedinto a test tube. Suitable and preferred flea saliva products for usewith an in vitro hypersensitivity test of the present invention are asdisclosed for a skin test of the present invention.

[0219] A second step of a preferred in vitro hypersensitivity test ofthe present invention comprises contacting the coated substrate with abody fluid, such as serum, plasma or whole blood, from an animalsusceptible to allergic dermatitis in such a manner as to allowantibodies contained in the body fluid that are capable of binding toectoparasite saliva products to bind to such products bound to thesubstrate to form immunocomplexes. Excess body fluid and antibodies arethen washed from the substrate. In a preferred embodiment in which IgEantibodies in the body fluid are to be measured, the body fluid can bepretreated to remove at least some of the other isotypes ofimmunoglobulin and/or other proteins, such as albumin, present in thefluid. Such removal can include, but is not limited to, contacting thebody fluid with a material, such a Protein G, to remove IgG antibodiesand/or affinity purifying the IgE antibodies from other components ofthe body fluid by exposing the fluid to, for example, Concanavalin A(Con-A).

[0220] A third step of a preferred in vitro hypersensitivity test of thepresent invention comprises contacting the immunocomplexes bound to thesubstrate with a compound capable of bi ding to the immunocomplexes,such as a secondary antibody or other compound that is capable ofbinding to the heavy chain of allergy-related antibodies produced byanimals allergic to ectoparasites, in such a manner that the compound(s)can bind to the immunocomplexes. Preferred binding compounds include,but are not limited to, secondary antibodies capable of binding to theheavy chain of IgE antibodies. Preferred animals to test are disclosedherein. Compounds capable of binding to immunocomplexes are usuallytagged with a label which enables the amount of compound bound to theantibody from the body fluid to be measured. Such labels include, butare not limited to, a radioactive label, an enzyme capable of producinga color reaction upon contact with a substrate, a fluorescent label, achemiluminescent label, a chromophoric label or a compound capable ofbeing bound by another compound. Preferred labels include, but are notlimited to, fluorescein, radioisotopes, alkaline phosphatases, biotin,avidin, or peroxidases.

[0221] A fourth step of a preferred in vitro hypersensitivity test ofthe present invention comprises measuring the amount of detectable labelbound to the solid substrate using techniques known to those of skill inthe art. It is within the scope of the present invention that the amountof antibody from the body fluid bound to the substrate can be determinedusing one or more layers of secondary antibodies or other bindingcompounds. For example, an untagged secondary antibody can be bound to aserum antibody and the untagged secondary antibody can then be bound bya tagged tertiary antibody.

[0222] A hypersensitive animal is identified by comparing the level ofimmunocomplex formation using samples of body fluid with the level ofimmunocomplex formation using control samples. An immunocomplex refersto a complex comprising an antibody and its ligand (i.e., antigen). Assuch, immunocomplexes form using positive control samples and do notform using negative control samples. As such, if a body fluid sampleresults in immunocomplex formation greater than or equal toimmunocomplex formation using a positive control sample, then the animalfrom which the fluid was taken is hypersensitive to the ectoparasitesaliva product bound to the substrate. Conversely, if a body fluidsample results in immunocomplex formation similar to immuno omplexformation using a negative control sample, then the animal from whichthe fluid was taken is not hypersensitive to the ectoparasite salivaproduct bound to the substrate.

[0223] One embodiment of the present invention is a kit useful foridentification of an animal susceptible to or having allergicdermatitis. As used herein, a suspect animal is an animal to be tested.A kit of the present invention comprises a formulation of the presentinvention and a means for determining if an animal is susceptible to orhas allergic dermatitis, in which the formulation is used to identifyanimals susceptible to or having allergic dermatitis. A means fordetermining if an animal is susceptible to or has allergic dermatitiscan include an in vivo or in vitro hypersensitivity test of the presentinvention as described in detail above. A kit of the present inventionfurther comprises at least one control solution such as those disclosedherein.

[0224] A preferred kit of the present invention comprises the elementsuseful for performing an immunoassay. A kit of the present invention cancomprise one or more experimental samples (i.e., formulations of thepresent invention) and one or more control samples bound to at least onepre-packed dipstick, and the necessary means for detecting immunocomplexformation (e.g., labelled secondary antibodies or other bindingcompounds and any necessary solutions needed to resolve such labels, asdescribed in detail above) between antibodies contained in the bodilyfluid of the animal being tested and the proteins bound to the dipstick.It is within the scope of the invention that the kit can comprise simplya formulation of the present invention and that the detecting means canbe provided in another way.

[0225] An alternative preferred kit of the present invention compriseselements useful for performing a skin test. A kit of the presentinvention can comprise at least one pre-packed syringe and needleapparatus containing one or more experimental samples and/or one or morecontrol samples.

[0226] It is within the scope of the present invention that two or moredifferent in vivo and/or in vitro tests can be used in combination fordiagnostic purposes. For example, the immediate hypersensitivity of ananimal to an ectoparasite saliva allergen can be tested using an invitro immunoabsorbent test capable of detecting IgE antibodies specificfor an ectoparasite saliva allergen in the animal's bodily fluid. Whilemost animals that display delayed hypersensitivity to an ectoparasitesaliva allergen also display immediate hypersensitivity to the allergen,a small number of animals that display delayed hypersensitivity to anallergen do not display immediate hypersensitivity to the allergen. Insuch cases, following negative results from the IgE-specific in vitrotest, the delayed hypersensitivity of the animal to an ectoparasitesaliva allergen can be tested using an in vivo test of the presentinvention.

[0227] Another aspect of the present invention includes treating animalssusceptible to or having allergic dermatitis, with a formulation of thepresent invention. According to the present invention, the termtreatment can refer to the regulation of a hypersensitive response by ananimal to bites from ectoparasites. Regulation can include, for example,immunomodulation of cells involved in the animal's hypersensitiveresponse or alteration of the ability of an ectoparasite to introduceallergens into an animal, for example by inhibiting the anti-coagulationactivity of a saliva enzyme, thereby impairing the ability of thearthropod to penetrate the dermis of an animal and feed.Immunomodulation can include modulating the activity of moleculestypically involved in an immune response (e.g., antibodies, antigens,major histocompatibility molecules (MHC) and molecules co-reactive withMHC molecules). In particular, immunomodulation refers to modulation ofantigen:antibody interactions resulting in inflammatory responses,immunosuppression, and immunotolerization of cells involved in ahypersensitive response. Immunosuppression refers to inhibiting animmune response by, for example, killing particular cells involved inthe immune response. Immunotolerization refers to inhibiting an immuneresponse by anergizing (i.e., diminishing reactivity of a T cell to anantigen) particular cells involved in the immune response. Suitable andpreferred ectoparasites against which to treat an animal are disclosedherein. A particularly preferred formulation of the present invention isused to treat FAD.

[0228] One embodiment of the present invention is a therapeuticcomposition that, when administered to an animal in an effective manner,is useful for immunomodulating the immune response of the animal (i.e.,immunomodulating the animal) so as to block (i.e., to inhibit, reduce orsubstantially prevent) a hypersensitive response by the animal uponsubsequent exposure to allergenic components transmitted through bitesfrom ectoparasites. Such a therapeutic composition is useful forimmunomodulating animals known to be hypersensitive to ectoparasitesaliva products and animals susceptible to hypersensitive responsesagainst ectoparasite saliva products.

[0229] One embodiment of the present invention is a therapeuticcomposition that includes de-sensitizing compounds capable of inhibitingan immune response to an ectoparasite saliva product of the presentinvention. Such de-sensitizing compounds include blocking compounds,toleragens and/or suppressor compounds. Blocking compounds comprisecompounds capable of modulating antigen:antibody interactions that canresult in inflammatory responses, toleragens are compounds capable ofimmunotolerizing an animal, and suppressor ccapounds are capable ofimmunosuppressing an animal. A de-sensitizing compound of the presentinvention can be soluble or membrane-bound. Membrane-boundde-sensitizing compounds can be associated with biomembranes, includingcells, liposomes, planar membranes or micelles. A soluble de-sensitizingcompound of the present invention is useful for: (1) inhibiting a Type Ihypersensitivity reaction by blocking IgE:antigen mediatedde-granulation of mast cells; (2) inhibiting a Type III hypersensitivityreaction by blocking IgG:antigen complex formation leading to complementdestruction of cells; and (3) inhibiting a Type IV hypersensitivityreaction by blocking T helper cell stimulation of cytokine secretion bymacrophages. A membrane-bound de-sensitizing compound of the presentinvention is useful for: (1) inhibiting a Type II hypersensitivityreaction by blocking IgG:antigen complex formation on the surface ofcells leading to complement destruction of cells; (2) inhibiting a TypeII hypersensitivity reaction by blocking IgG regulated signaltransduction in immune cells; and (3) inhibiting a Type IVhypersensitivity reaction by blocking T cytotoxic cell killing ofantigen-bearing cells.

[0230] A de-sensitizing compound of the present invention can also becovalently linked to a ligand molecule capable of targeting thede-sensitizing compound to a specific cell involved in a hypersensitiveresponse to ectoparasite saliva products. Appropriate ligands with whichto link a de-sensitizing compound include, for example, at least aportion of an immunoglobulin molecule, cytokines, lectins, heterologousallergens, CD8 molecules or major histocompatibility molecules (e.g.,MHC class I or MHC class II molecules). Preferred portions ofimmunoglobulin molecules to link to a de-sensitizing compound includevariable regions capable of binding to immune cell specific surfacemolecules and constant regions capable of binding to Fc receptors onimmune cells, in particular IgE constant regions. Preferred CD8molecules include at least the extracellular functional domains of the αchain of CD8. An immune cell refers to a cell involved in an immuneresponse, in particular, cells having MHC class I or MHC class IImolecules. Preferred immune cells include antigen presenting cells, Tcells and B cells.

[0231] In one embodiment, a therapeutic composition of the presentinvention includes ectoparasite saliva products of the presentinvention, or mimetopes thereof. Preferred therapeutic compositionsinclude formulations comprising ectoparasite saliva extracts or at leastone ectoparasite saliva product (preferably protein) of the presentinvention or mimetopes thereof.

[0232] Suitable therapeutic compositions of the present invention fortreating flea allergy dermatitis include flea saliva extracts and otherformulations including at least one flea saliva product, preferably aprotein, or a mimetope thereof. Preferred therapeutic compositionsinclude FS-1, FS-2 and/or FS-3 as well as at least a portion of at leastone flea saliva product that can be isolated from FS-1, FS-2 and/orFS-3. As such, preferred formulations for use as therapeuticcompositions include FS-1, FS-2, FS-3, and/or at least a portion of oneor more of the proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE,fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2,fspM1, fspM2, fspN1, fspN2 and fspN3, or homologues thereof. A morepreferred flea saliva extract for use as a therapeutic compositionsincludes FS-1, FS-2, FS-3, and/or at least a portion cf one or more ofthe proteins fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2,fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3. A yet morepreferred flea saliva extract for use as a therapeutic compositionsincludes FS-1, FS-2, and/or at least a portion of one or more of theproteins fspG1, fspG2, fspG3, fspH, fspM1, fspM2, fspN1, fspN2 andfspN3.

[0233] In another embodiment, a therapeutic composition can includeectoparasite products of the present invention associated with asuitable excipient. A therapeutic composition of the present inventioncan be formulated in an excipient that the animal to be treated cantolerate. Preferred excipients are capable of maintaining a product ofthe present invention in a form that is capable of being bound by cellsinvolved in an allergic response in an animal such that the cells arestimulated to initiate or enhance an immune response. Examples of suchexcipients include water, saline, Ringer's solution, dextrose solution,Hank's solution, and other aqueous physiologically balanced saltsolutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyloleate, or triglycerides may also be used. Other useful formulationsinclude suspensions containing viscosity enhancing agents, such assodium carboxymethylcellulose, sorbitol, or dextran. Excipients can alsocontain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, m- or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, preservatives, etc., towhich sterile water or saline can be added prior to administration.

[0234] In another embodiment, a therapeutic composition of the presentinvention can also comprise a carrier or adjuvant, although it is to beappreciated that an advantage of saliva products of the presentinvention is that adjuvants and/or carriers are not required foradministration. Adjuvants are typically substances that generallyenhance the immune response of an animal to a specific antigen. Suitableadjuvants include, but are not limited to, Freund's adjuvant; otherbacterial cell wall components; aluminum-based salts; calcium-basedsalts; silica; polynucleotides; toxoids; serum proteins; viral coatproteins; other bacterial-derived preparations; gamma interferon; blockcopolymer adjuvants, such as Hunter's Titermax adjuvant (Vaxcel™, Inc.Norcross, Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research,Inc., Hamilton, Mont.); and saponins and their derivatives, such as QuilA (available from Superfos Biosector A/S, Denmark).

[0235] Carriers are typically compounds that increase the half-life of atherapeutic composition in the treated animal. Suitable carriersinclude, but are not limited to, polymeric controlled releaseformulations, biodegradable implants, liposomes, bacteria, viruses,oils, esters, and glycols.

[0236] One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a therapeuticcomposition of the present invention into the bloodstream of an animal.Suitable controlled release formulations include, but are not limitedto, biocompatible (including biodegradable) polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ.

[0237] The present invention also includes a recombinant virus particletherapeutic composition. Such a composition includes a recombinantmolecule of the present invention that is packaged in a viral coat andthat can be expressed in an animal after administration. Preferably, therecombinant molecule is packaging-deficient. A number of recombinantvirus particles can be used, including, but not limited to, those basedon alphaviruses, poxviruses, adenoviruses, herpesviruses, andretroviruses. Preferred recombinant particle viruses are those based onalphaviruses (such as Sindbis virus), herpesviruses and poxviruses.Methods to produce and use recombinant virus particle vaccines aredisclosed in U.S. patent application Ser. No. 08/015/414, filed Feb. 8,1993, entitled “Recombinant Virus Particle Vaccines”, which isincorporated by reference herein in its entirety.

[0238] When administered to an animal, a recombinant virus particletherapeutic composition of the present invention infects cells withinthe immunized animal and directs the production of a protective proteinor RNA nucleic acid molecule that is capable of protecting the animalfrom allergic dermatitis caused by the bites of ectoparasites. Forexample, a recombinant virus particle comprising a nucleic acid moleculeencoding one or more ectoparasite saliva protein of the presentinvention is administered according to a protocol that results in thetolerization of an animal against ectoparasite saliva allergens.

[0239] Therapeutic compositions of the present invention can besterilized by conventional methods which do not result in proteindegradation (e.g., filtration) and/or lyophilized.

[0240] A therapeutic composition of the present invention can beadministered to any animal susceptible to ectoparasite infestation asherein described. Acceptable protocols by which to administertherapeutic compositions of the present invention in an effective mannercan vary according to individual dose size, number of doses, frequencyof dose administration, and mode of administration. Determination ofsuch protocols can be accomplished by those skilled in the art. Aneffective dose refers to a dose capable of treating an animal againsthypersensitivity to ectoparasite saliva allergens. Effective doses canvary depending upon, for example, the therapeutic composition used, thearthropod from which the composition was derived, and the size and typeof the recipient animal. Effective doses to immunomodulate an animalagainst ectoparasite saliva allergens include doses administered overtime that are capable of alleviating a hypersensitive response by ananimal to ectoparasite saliva allergens. For example, a first tolerizingdose can comprise an amount of a therapeutic composition of the presentinvention that causes a minimal hypersensitive response whenadministered to a hypersensitive animal. A second tolerizing dose cancomprise a greater amount of the same therapeutic composition than thefirst dose. Effective tolerizing doses can comprise increasingconcentrations of the therapeutic composition necessary to tolerize ananimal such that the animal does not have a hypersensitive response tothe bite of an ectoparasite. An effective dose to desensitize an animalcan comprise a concentration of a therapeutic composition of the presentinvention sufficient to block an animal from having a hypersensitiveresponse to the bite of an ectoparasite. Effective desensitizing dosescan include repeated doses having concentrations of a therapeuticcomposition that cause a minimal hypersensitive response whenadministered to a hypersensitive animal.

[0241] A suitable single dose is a dose that is capable of treating ananimal against hypersensitivity to ectoparasite saliva allergens whenadministered one or more times over a suitable time period. For example,a preferred single dose of an ectoparasite saliva product, or mimetopetherapeutic composition is from about 0.5 ng to about 1 g of thetherapeutic composition per kilogram body weight of the animal. Furthertreatments with the therapeutic composition can be administered fromabout 1 hour to 1 year after the original administration. Furthertreatments with the therapeutic composition preferably are administeredwhen the animal is no longer protected from hypersensitive responses toectoparasite. Particular administration doses and schedules can bedeveloped by one of skill in the art based upon the parameters discussedabove. Modes of administration can include, but are not limited to,subcutaneous, intradermal, intravenous, nasal, oral, transdermal andintramuscular routes.

[0242] A therapeutic composition of the present invention can be used inconjunction with other compounds capable of modifying an animal'shypersensitivity to ectoparasite bites. For example, an animal can betreated with compounds capable of modifying the function of a cellinvolved in a hypersensitive response, compounds that reduce allergicreactions, such as by systemic agents or anti-inflammatory agents (e.g.,anti-histamines, anti-steroid reagents, anti-inflammatory reagents andreagents that drive immunoglobulin heavy chain class switching from IgEto IgG). Suitable compounds useful for modifying the function of a cellinvolved in a hypersensitive response include, but are not limited to,antihistamines, cromolyn sodium, theophylline, cyclosporin A, adrenalin,cortisone, compounds capable of regulating cellular signal transduction,compounds capable of regulating adenosine 3′,5′-cyclic phosphate (cAMP)activity, and compounds that block IgE activity, such as peptides fromIgE or IgE specific Fc receptors, antibodies specific for peptides fromIgE or IgE-specific Fc receptors, or antibodies capable of blockingbinding of IgE to Fc receptors.

[0243] Another aspect of the present invention includes a method forprescribing treatment for animals susceptible to or having allergicdermatitis, using a formulation of the present invention. A preferredmethod for prescribing treatment for flea allergy dermatitis, forexample, comprises: (1) intradermally injecting into an animal at onesite an effective amount of a formulation containing at least one fleasaliva antigen of the present invention, or a mimetope thereof (suitableand preferred formulations are disclosed herein); (2) intradermallyinjecting into the animal at a second site an effective amount of acontrol solution; (3) evaluating if the animal has flea allergydermatitis by measuring and comparing the wheal size resulting frominjection of the formulation with the wheal size resulting frominjection of the control solution; and (4) prescribing a treatment forthe flea allergy dermatitis.

[0244] An alternative preferred method for prescribing treatment forflea allergy dermatitis comprises: (1) contacting a first portion of asample of bodily fluid obtained from an animal to be tested with aneffective amount of a formulation containing at least one flea salivaantigen, or a mimetope thereof (suitable and preferred formulations aredisclosed herein) to form a first immunocomplex solution; (2) contactinga positive control antibody to form a second immunocomplex solution; (3)evaluating if the animal has flea allergy dermatitis by measuring andcomparing the amount of immunocomplex formation in the first and secondimmunocomplex solutions; and (4) prescribing a treatment for the fleaallergy dermatitis. It is to be noted that similar methods can be usedto prescribe treatment for allergies caused by other ectoparasites usingectoparasite saliva product formulations as disclosed herein.

[0245] Another aspect of the present invention includes a method formonitoring animals susceptible to or having allergic dermatitis, using aformulation of the present invention. In vivo and in vitro tests of thepresent invention can be used to test animals for allergic dermatitisprior to and following any treatment for allergic dermatitis. Apreferred method to monitor treatment of flea allergy dermatitis (whichcan also be adapted to monitor treatment of other ectoparasiteallergies) comprises: (1) intradermally injecting an animal at one sitewith an effective amount of a formulation containing at least one fleasaliva product, or a mimetope thereof (suitable and preferredformulations are disclosed herein); (2) intradermally injecting aneffective amount of a control solution into the animal at a second site;and (3) determining if the animal is desensitized to flea salivaantigens by measuring and comparing the wheal size resulting frominjection of the formulation with the wheal size resulting frominjection of the control solution.

[0246] An alternative preferred method to monitor treatment of fleaallergy dermatitis (which can be adapted to monitor treatments of otherectoparasite allergies) comprises: (1) contacting a first portion of asample of bodily fluid obtained from an animal to be tested with aneffective amount of a formulation containing at least one flea salivaproduct or mimetope thereof (suitable and preferred formulations aredisclosed herein) to form a first immunocomplex solution; (2) contactinga positive control antibody to form a second immunocomplex solution; and(3) determining if the animaal is desensitized to flea saliva antigensby measuring and comparing the amount of immunocomplex formation in thefirst and second immunocomplex solutions.

[0247] The present invention also includes antibodies capable ofselectively binding to an ectoparasite saliva product, or mimetopethereof. Such an antibody is herein referred to as an anti-ectoparasitesaliva product antibody. As used herein, the term “selectively binds to”refers to the ability of such an antibody to preferentially bind toectoparasite saliva products and mimetopes thereof. In particular, thepresent invention includes antibodies capable of selectively binding toflea saliva products. Binding can be measured using a variety of methodsknown to those skilled in the art including immunoblot assays,immunoprecipitation assays, enzyme immunoassays (e.g., ELISA),radioimmunoassays, immunofluorescent antibody assays and immunoelectronmicroscopy; see, for example, Sambrook et al., ibid.

[0248] Antibodies of the present invention can be either polyclonal ormonoclonal antibodies. Antibodies of the present invention includefunctional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies,that are capable of selectively binding to at least one of the epitopesof the protein or mimetope used to obtain the antibodies. Preferredantibodies are raised in response to ectoparasite saliva proteins, ormimetopes thereof. More preferred antibodies are raised in response toat least one ectoparasite saliva protein, or mimetope thereof, having atleast a portion of an ectoparasite saliva protein eluted from acollection means of the present invention. Even more preferredantibodies are raised in response to at least one flea saliva product,or homologues thereof (e.g., saliva products of other ectoparasites),contained in the saliva extracts FS-1, FS-2 and/or FS-3. More preferredectoparasite saliva proteins against which to raise an antibody includesat least a portion of one or more of the proteins fspA, fspB, fspC1,fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1,fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3, orhomologues thereof. Preferably, an antibody of the present invention hasa single site binding affinity of from about 10³ M⁻¹ to about 10¹² M⁻¹for a flea saliva product of the present invention.

[0249] A preferred method to produce antibodies of the present inventionincludes administering to an animal an effective amount of anectoparasite saliva product or mimetope thereof to produce the antibodyand recovering the antibodies. Antibodies raised against definedproducts or mimetopes can be advantageous because such antibodies arenot substantially contaminated with antibodies against other substancesthat might otherwise cause interference in a diagnostic assay or sideeffects if used in a therapeutic composition.

[0250] Antibodies of the present invention have a variety of potentialuses that are within tne scope of the present invention. For example,such antibodies can be used (a) as vaccines to passively immunize ananimal in order to protect the animal from allergic dermatitis, (b) aspositive controls in test kits, and/or (c) as tools to recover desiredectoparasite saliva products from a mixture of proteins and othercontaminants.

[0251] The following examples are provided for the purposes ofillustration and are not intended to limit the scope of the presentinvention.

EXAMPLES Example 1

[0252] This example describes the collection of flea saliva proteinsusing a saliva collection apparatus of the present invention.

[0253] A saliva collection apparatus was prepared as follows. Referringto FIGS. 4A and 4B, a humidifying means (34) comprising about 4 piecesof VWR blotting pads #320 (VWR, Denver, Colo.) was prepared that fit theinner diameter (about 47 mm in diameter) of a chamber (6) of a salivacollection apparatus (2). The blotting pads were pre-wetted using asufficient amount of pre-wetting solution (sterile water containing 50units/ml penicillin and 50 μg/ml streptomycin, available from Sigma, St.Louis, Mo.) such that the blotting pads were damp but not dripping wet.The pre-wetted filters (34) were placed inside the bottom end (22) ofthe chamber (6) of the saliva collection apparatus (2) such that thefilter paper sat immediately inside the bottom end (22) of the chamber(6).

[0254] A collection means (24) comprising a Durapore™ membrane(available from Millipore, Bedford, Mass.) was cut to fit the outerdiameter (about 48 mm in diameter) of the chamber (6) of the salivacollection apparatus (2). The Durapore™ membrane was pre-wetted usingthe pre-wetting solution described above. The Durapore™ membrane (24)was placed immediately outside the bottom end (22) of the chamber (6)such that the Durapore™ membrane (24) contacted the outer rim of thebottom end (22) of the chamber (6) and also contacted the damp filterpaper. A barrier means comprising a piece of stretched Parafilm™ (28)(available from American National Can™, Greenwich, Conn.) was stretchedover the collection means (24) and bottom end (22) of the chamber (6)and up the outer wall (30) of the chamber (6). A rubber seal (32) (i.e.,an O-ring) was placed over the Parafilm™ (28) thereby further securingthe Parafilm™ (28) across the collection means (24) and to the outerwall (30) and to seal in the chamber (6) environment.

[0255] The collection apparatus (2) was preassembled and then the topend (20) of the chamber (6) was attached to an artificial feeding systemcapable of acting as a source of heat and humidity such as thatdescribed by Wade et al., (ibid.). The artificial feeding systemcomprised a large plexiglass box (40 cm×40 cm×40 cm) dividedhorizontally into an upper compartment and a lower compartment by aplexiglass shelf having holes drilled through. A collection apparatus(2) was inserted into a hole such that the chamber (6) of the apparatus(2) was located above the shelf in the upper compartment and the housing(4) was located below the shelf in the lower compartment. The apparatus(2) was secured to the shelf by attaching a rubber band attached tometal hooks placed in the shelf. Any open holes in the shelf were closedoff using rubber stoppers to isolate the environment within the uppercompartment from the environment within the lower compartment. The uppercompartment contained two trays of water, a fan and a heating block. Thetrays of water were placed such that the fan faced the trays. While theapparatus (2) was maintained in the artificial feeding system, the fanwas blown continuously thereby circulating heat and humidity throughoutthe upper compartment and the chamber (6) of the collection apparatus(2). As such, the relative humidity within the chamber (6) wasmaintained at about 94% humidity and the temperature was maintained atabout 37° C.

[0256] About 3,000 to 5,000 newly emerged unfed Ctenocephalides felisfleas were added to the housing (4) of the collection apparatus (2). Thefleas were first collected in a 20 gallon glass aquarium. The fleas werethen transferred to the housing (4) of a collection apparatus (2) byplacing the end of the housing (4) having the nylon mesh of the exchangemeans (16) on top of a vacuum chamber and aspirating the fleas from theaquarium into the housing (4) through a tygon tubing. The housing (4)was then covered with the nylon mesh of the retaining means (18) tosecure the fleas within the housing (4). The bottom end (22) of thechamber (6) was then placed on the housing (4) such that the Parafilm™(28) and the nylon mesh of the retaining means (18) were juxtaposed.When the collection apparatus (2) was attached to the artificial feedingsystem, the ambient temperature within the housing (4) was maintained atabout 27° C. while the ambient temperature of the chamber (6) wasmaintained at about 37° C. The relative humidity of the housing (4) wasmaintained at about 50% by cl sing the lower compartment with theplexiglass shelving.

[0257] In one experiment, flea saliva products were collected on aDurapore™ membrane (24) and visualized by immersing the membrane in 0.1%Coomassie blue stain for 20 minutes, destaining the membrane in 50%methanol and air drying the membrane. Proteins deposited on the membranewere detected by their blue color.

[0258] In another experiment, flea saliva products were collected for 0through 24 hours, 24 through 72 hours, and 72 through 120 hours afterloading fleas into the collection apparatus. At 24 hours, 72 hours and120 hours, the Durapore™ membrane (24) attached to the collectionapparatus (2) was removed and a new pre-wetted Durapore™ membrane (24)was attached to the same apparatus. The blotting pads were re-wettedusing the pre-wetting solution described above when the new Durapore™membrane (24) was replaced. Flea saliva products were extracted from theDurapore™ membrane (24) by soaking each membrane from each time pointseparately in a solvent comprising 50% acetonitrile with 1% TFAovernight at room temperature with stirring to obtain a flea salivaproduct mixture comprising flea saliva products that had eluted into thesolvent. The mixture containing the flea saliva products was recoveredand lyophilized until dry to form a pellet. The amount andcharacteristics of flea saliva proteins eluted from each Durapore™membrane from each time point was determined by reducing 14%Tris-glycine SDS-PAGE using techniques similar to those described bySambrook et al., ibid. The resultant protein pattern was visualized bystaining the gel with Coomassie blue stain using techniques as describedabove. The amount of saliva proteins collected on the membranesdecreased when the fleas had been in the collection apparatus for morethan 72 hours.

Example 2

[0259] Standard procedures to collect FS-1, FS-2 and FS-3 flea salivaextracts of the present invention were performed as follows. Flea salivaproducts were collected for 72 hours on collection membranes using themethod described in Example 1, except that for flea saliva extract FS-3,the collection membrane was DE-81 chromatography paper, available fromWhatman, Inc., Clifton, N.J.

[0260] A. Flea Saliva Extracts FS-1 and FS-2:

[0261] Flea saliva products were extracted from the Durapore™ membrane(24) by soaking each membrane from each time point separately in a firstsolvent comprising 50% acetonitrile with 0.1% TFA for 8 hours. The firstmixture containing the eluted flea saliva products was recovered andlyophilized until dry, thereby forming a first pellet. The samemembranes were then soaked in a second solvent comprising 50%acetonitrile with 1% TFA overnight at room temperature with stirring toobtain a flea saliva product mixture comprising flea saliva productsthat had eluted into the second solvent. The second mixture wasrecovered from this second extraction and lyophilized until dry to forma second pellet.

[0262] The two pellets recovered from the two lyophilization steps weremixed with a third solvent comprising 12.8% acetonitrile and flea salivaproducts solubilized in the solvent were recovered. The non-solubilizedmaterial was mixed again with 12.8% acetonitrile and additional fleasaliva products solubilized in the solvent were recovered. The twomixtures were combined to obtain the extract FS-1.

[0263] The non-solubilized material remaining after the secondsolubilization step was then mixed with 50% acetonitrile whichsolubilized the remaining material to obtain the extract FS-2.

[0264] The amount and characteristics of flea saliva proteins containedin the FS-1 and FS-2 flea saliva extracts obtained in at least oneexperiment were determined according to the following method. Eachextract was concentrated by evaporation under vacuum and evaluated byreducing 16% Tris-glycine SDS-PAGE using techniques similar to thosedescribed by Sambrook et al., ibid. Using such standard procedures,about 10 μg of FS-1 or FS-2 eluted from the Durapore™ membrane wasloaded onto a 16% Tris-glycine polyacrylamide gel and subjected toelectrophoresis under reducing conditions. The gel was stained withCoomassie blue and dried.

[0265] The results are shown in FIG. 1B. FS-1 is shown in lane 13 ofFIG. 1B and FS-2 is shown in lanes 14 and 15 of FIG. 1B. FS-1 was foundto contain proteins estimated to have the following molecular weights: 9kD, 11 kD, 12 kD, 15 kD, 22 kD, 48 kD, 50 kD, 53 kD, 80 kD, 124 kD, 130kD, 189 kD and 201 kD. Those proteins of 80 kD and above were muchfainter than the lower molecular weight bands. FS-2 was found to containproteins having the following molecular weights: 47 kD, 49 kD, 52 kD, 57kD, 64 kD, 71 kD, 88 kD, 96 kD, 97 kD, 130 kD, 161 kD, 175 kD, 189 kD,222 kD, 235 kD and 302 kD. The bands at 47 kD, 49 kD and 52 kD were moreprominent than the bands having higher molecular weights. The resultssuggest that a substantial portion of the protein contained in FS-2 isfspN1, fspN2 and/or fspN3.

[0266] Protein concentrations were measured using a Bio-Rad Bradfordassay (available from Bio-Rad, Hercules, Calif.). The results indicatethat about 750 μg of protein can be collected in about 3.66×10⁷ fleahours (5.08×10⁵ fleas for 72 hours) in an FS-1 extract and about 2.35 mgof protein can be collected in about 3.66×10⁷ flea hours in an FS-2extract.

[0267] B. Flea Saliva Extract FS-3:

[0268] Flea saliva products to produce FS-3 flea saliva extract werecollected in a manner similar to the method by which FS-1 and FS-2 werecollected, except that the collection membrane (24) was DE-81chromatography paper. Flea saliva products were extracted from the DE-81membrane by soaking each membrane from each time point separately in asolvent comprising 1M NaCl in phosphate buffered saline for about 8hours. The products were recovered from the solvent using standardtechniques, such as disclosed for FS-1 and FS-2.

[0269] Analysis of an FS-3 flea saliva extract indicated that FS-3appeared to contain proteins such as those found in FS-1 and FS-2, atleast based on 1-dimensional gel electrophoresis. The SDS-PAGE patternof FS-3, for example, was very similar to that of FS-1 except that thereappeared to be increased quantities of the higher molecular weightproteins in the FS-3 extract. FS-3 flea saliva extract was also shown tohave anti-coagulation activity, using techniques standard in the art;see, for example, Dunwiddle et al., 1991, Thrombosis Research 64,787-794; Ribeiro et al., 1990, Comp. Biochem. Physiol. 95, 215-218;Ribeiro et al., 1990, Br. J. Pharmacol. 101, 932-936; Ribeiro et al.,1987, Exper. Parasitol. 64, 347-353; Cupp et al., 1994, Am. J. Trop.Med. Hyg. 50, 241-246; Garcia et al., 1994, Exper. Parasitol. 78,287-293. The FS-3 extract was also shown to exhibit acid phosphataseactivity, using techniques standard in the art, such as those suppliedby Sigma, St. Louis, Mo., with the Sigma acid phosphtase assay kit.

Example 3

[0270] This example describes the characterization by HPLC of fleasaliva proteins collected using a saliva collection apparatus of thepresent invention.

[0271] An FS-1 flea saliva extract was collected as described in Example2 from about 140,000 fleas for 72 hours. Proteins contained in FS-1 wereresolved using standard procedures of high pressure liquidchromatography (HPLC). Specifically, the proteins were passed over a 15cm×0.46 cm C4 column using a gradient from 0.1% TFA in water (Solvent A)to 0.085% TFA in 90% CH₃CN (Solvent B) at a flow rate of 0.8 ml perminute. Thus, the gradient was about 5.6% Solvent B at 15 minutes andabout 100% Solvent B at 75 minutes.

[0272] The results are shown in FIG. 2. About 14 major protein fractionswere resolved. The recovery for each peak was about 5 μg to 10 μg ofprotein per peak. The peaks were labelled peak A, peak B, peak C, peakD, peak E, peak F, peak G, peak H, peak I, peak J, peak K, peak L, peakM and peak N, as shown in FIG. 2, and represent, respectively, proteinformulations fspA, fspB, fspC1 and fspC2, fspD1 and fspD2, fspE, fspF,fspG1, fspG2, fspG3, fspH, fspI, fspJ1 and fspJ2, fspK, fspL1 and fspL2,fspM1and fspM2, and fspN1, fspN2 and fspN3.

[0273] Samples from each HPLC peak were resolved by Tris GlycineSDS-PAGE gels using the method described in Example 1. The results areshown in FIGS. 1A, 1B and 1C. The proteins shown in FIGS. 1A and 1B wereresolved on 16% Tris Glycine SDS-PAGE gels and the proteins shown inFIG. 1C were resolved on a 14% Tris Glycine SDS-PAGE gel. Proteinmarkers are shown in lane 1 of FIG. 1A, lane 2 of FIG. 1B and lane 1 ofFIG. 1C. The additional lanes show saliva formulation samples asfollows: Lane Fraction(s) Fs-( ) FIG. 1A  1) — Mol. Wt. Std.  2) 10 — 3) 11-13 A  4) 14 B  5) 15 B  6) 16 C1  7) 17 C2  8) 18 D1  9) 19 D110) 20 D2 11) 21 D2 12) 22 E 13) 23 F 14) 24 G 15) 25 G FIG. 1B  1)26-27 G  2) — Mol. Wt. Std.  3) 28 H  4) 29-30 I  5) 31 J  6) 32 K  7)33 K  8) 34 L  9) 35 M1 10) 36-37 M1 11) 38 M1 12) 39-50 M2 13) — FS-114) — FS-2 15) — FS-2 FIG. 1C  1) — Mol. Wt. Std.  2) 56-68 N

[0274] Referring to FIG. 1A, the following flea saliva proteins(referred to as bands) were observed: a prominent band of about 10 kD inpeak A and peak B samples; a prominent band of about 6 kD and a lessprominent band of 9 kD in a peak C sample referred to as C1; a prominentband of about 7 kD in a peak C sample referred to as C2; a prominentband of about 7 kD and a less prominent band of 8 kD in a peak D samplereferred to as Dl; a prominent band of about 8 kD and a less prominentband of 9 kD in a peak D sample referred to as D2; a prominent band of 8kD and a less prominent band of about 7 kD in peaks E and F samples; anda prominent band of about 9 kD, and less prominent bands of about 7 kDand 10 kD in a peak G sample. Referring to FIG. 1B, the following fleasaliva proteins were observed: a prominent band of about 9 kD and a lessprominent band of about 12 kD in a peak H sample; a prominent band ofabout 21 kD, and less prominent bands of about 7 kD, 9 kD, 12 kD, 14 kD,and 70 kD in a peak I sample; prominent bands of about 14 kD and 21 kD,and less prominent bands of about 11 kD and 17 kD in a peak J sample;prominent bands of about 14 kD and 15 kD and less prominent bands ofabout 12 kD, 18 kD and 21 kD in a peak K sample; a prominent band ofabout 15 kD in a peak L sample; prominent bands of about 11 kD, 12 kDand 21 kD and less prominent bands of about 15 kD, 17 kD, 22 kD and 37kD in a peak M sample referred to as M1; and a prominent band of about36 kD and less prominent bands of about 11 kD, 21 kD and 22 kD in a peakM sample referred to as M2. Referring to FIG. 1C, prominent bands ofabout 42 kD, 43 kD and 44 kD and a less prominent band of about 32 kDwere detected in a peak N sample.

Example 4

[0275] This example describes the amino acid sequence analysis of theisolated and HPLC purified flea saliva proteins.

[0276] Amino (N-) terminal amino acid sequencing analysis was performedon several of the HPLC-separated flea saliva proteins described inExample 3 using standard procedures known to those in the art (see, forexample, Geisow et al., 1989, in Protein Sequencing: A PracticalApproach, J B C Findlay and M J Geisow (eds.), IRL Press, Oxford,England, pp. 85-98; Hewick et al., 1981, J. Biol. Chem., Vol. 256, pp.7990-7997).

[0277] The N-terminal partial amino acid sequence of flea saliva proteinfspA, which migrated as Peak A in FIG. 2, was determined to be Y G K Q YS E K G G R G Q R H Q I L K K G K Q Y S           S K       I     L D LS R

[0278] as represented in standard single letter code. This N-terminalpartial amino acid sequence of fspA is denoted SEQ ID NO: 1. It shouldbe noted that there was heterogeneity in several positions which mayrepresent sequence errors (i.e., misidentification of amino acids) orallelic variations in the flea population from which the saliva proteinswere collected. There was an apparently equal likelihood of finding anyone of the alternative amino acids at the indicated positions.

[0279] The N-terminal partial amino acid sequence of flea saliva proteinfspB, which migrated as Peak B in FIG. 2, was determined to be S/Q G K QY S E X G/S K, denoted SEQ ID NO: 27. This amino acid sequence wasessentially the same, or at least a subset of, the N-terminal amino acidsequence obtained from flea saliva protein fspA.

[0280] Sequence analysis of Peak G proteins indicated the presence ofthree proteins in that peak, referred to herein as fspG1, fspG2 andfspG3. Flea saliva protein fspG1, having a molecular weight of about 9kD, had an N-terminal partial amino acid sequence of D R R V S K,denoted SEQ ID NO: 28. This N-terminal amino acid sequence is the sameas that for fspH, as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5 and SEQ ID NO: 14. Flea saliva protein fspG2, having amolecular weight of about 7 kD, had an N-terminal partial amino acidseq-uence of S K M V T E K X K S G G N N P S T K E V S I P, denoted SEQID NO: 29. Flea saliva protein fspG3, having a molecular weight of about6 kD, had an N-terminal partial amino acid sequence of E V S I P S G K LT I E D F X I G N H Q, denoted SEQ ID NO: 30. A comparison of SEQ ID NO:30 with SEQ ID NO: 29 indicates that fspG3 may be a proteolyticdegradation product of fspG2, as the last five amino acids of fspG2 areidentical with those at the N-terminus of fspG3.

[0281] The N-terminal partial amino acid sequence of flea saliva proteinfspH, which migrated as Peak H in FIG. 2, was determined to be D R R V SK T X Q S G G K I Q S E X Q V V I K S G Q H/Y I L E N Y X S D G R,denoted herein as SEQ ID NO: 14. Histidine and tyrosine were equallylikely at amino acid position 27.

[0282] Flea saliva protein fspH was also subjected to proteolyticcleavage in order to obtain internal amino acid sequence data.Specifically, fspH was cleaved with Endoproteinase Asp-N (available fromBoehringer Mannheim Biochemica, Indianapolis, Ind.) using methodsstandard in the art. The digested protein was then resolved by HPLCusing the method described by Stone et al. (ibid.). The resultant HPLCprofile is shown in FIG. 3. Three proteolytic fragments were isolated,that are referred to herein as fspHe, fspHh and fspHj.

[0283] The N-terminal partial amino acid sequence of fspHe wasdetermined to be D S K H C Y C E A P Y S, also denoted SEQ ID NO: 3. TheN-terminal partial amino acid sequence of fspHh was determined to be D GR N N N N P C H L F C M R E C R S G N G G C G N G G R T R P D S K H C,also denoted SEQ ID NO: 4. The N-terminal partial amino acid sequence offspHj was determined to be D R R V S K T C Q S G, also denoted SEQ IDNO: 5. Comparison of SEQ ID NO: 5 to SEQ ID NO: 14 indicated that fspHjwas the N-terminal fragment of fspH.

[0284] By aligning SEQ ID NO: 14, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ IDNO: 5, the following amino acid sequence was deduced, starting at theN-terminus of fspH: D R R V S K T C Q S G G K I Q S E X Q V V I K S G QH/Y I L E N Y X S D G R N N N N P C H L F C M R E C R S G N G G C G N GG R T R P D S K H C Y C E A P Y S. This amino acid sequence is denotedSEQ ID NO: 2 and is believed to represent most of fspH since themolecular weight of a protein having this sequence is about 8600 kD.

[0285] The N-terminal partial amino acid sequence of flea saliva proteinfspI, which migrated as Peak I in FIG. 2, was determined to be E D I W KV N K K X T S G G K N Q D R K L D Q I I Q K G Q Q V X X Q N X X K,denoted herein as SEQ ID NO: 6.

[0286] Sequence analysis of Peak J proteins indicated the presence oftwo proteins in that peak, referred to herein as fspJ1 and fspJ2. TheN-terminal partial amino acid sequence of flea saliva protein fspJ1 wasdetermined to be N S H E P G N T R K I R E V M D K L R K Q H P, denotedherein as SEQ ID NO: 7. The N-terminal partial amino acid sequence offlea saliva protein fspJ2 was determined to be E I K R N S H E P G N T RK I R E V M D K L R K Q H P, denoted herein as SEQ ID NO: 8. Theproteins represented by SEQ ID NO: 7 and SEQ ID NO: 8 were notseparately resolved by SDS-PAGE as described in Example 1. Comparison ofSEQ ID NO: 7 and SEQ ID NO: 8 suggest that fspJ1 may be a truncatedversion of fspJ2, in that the N-terminal partial amino acid sequence offspJ1 appears to be very similar to that of fspJ2 except that fspJ1lacks the first 4 amino acids found at the N-terminus of fspJ2.

[0287] Sequence analysis of Peak L proteins indicated the presence oftwo proteins in that peak, referred to herein as fspL1 and fspL2. Thatthere were two proteins, namely fspL1 and fspL2, was shown by subjectingpeak L to C4 reverse phase chromatography using 0.13% heptafluorobutyricacid (Solvent A) and 0.1% heptafluorobutyric acid in 90% acetonitrile(Solvent B) in the following gradient format: an 80 minute gradient from30% Solvent B to 70% Solvent B. The N-terminal partial amino acidsequence of the HPLC-separated fspL1 was determined to be N D K E P G NT R K I R E V M D K L R K Q A Q P R T D G Q R P K T X I M, also denotedSEQ ID NO: 9. The N-terminal partial amino acid sequence for fspL2 wasdetermined to be X L X R N D K E P G N T R K I R E V M D K, also denotedSEQ ID NO: 10. A comparison of SEQ ID NO: 9 and SEQ ID NO: 10 indicatesthat fspL1 and fspL2 are similar proteins, except that fspL1 is 4 aminoacids shorter than fspL2 at the N-terminus.

[0288] Resolution of proteins contained in Peak N by SDS-PAGE asdescribed in Example 3 revealed 3 distinct bands. The bands were denotedflea saliva proteins fspN1, fspN2 and fspN3. The N-terminal partialamino acid sequence of fspN1 was determined to be N D E L K F V F V M AK, also denoted SEQ ID NO: 11. The N-terminal partial amino acidsequence of fspN2 was determined to be X D E L K F V F V M A K G P S X QA X D Y P C, also denoted SEQ ID NO: 12. The N-terminal partial aminoacid sequence of fspN3 was determined to be E L K F V F A T A R G M S HT P C D Y P, also denoted SEQ ID NO: 13. Comparison of SEQ ID NO: 11 andSEQ ID NO: 12 suggests that fspN1 and fspN2 share the same N-terminalsequence. Since fspN1 and fspN2 migrate differently when subjected toSDS-PAGE, however, the two proteins are likely to be differenthomologues, possibly due to one protein having a longer C-terminaldomain and/or due to post-translational modification(s). Comparison ofSEQ ID NO: 13 to SEQ ID NO: 11 and SEQ ID NO: 12 suggests that fspN3 maybe a homologue of fspN1 and fspN2 with internal sequence variations.

[0289] Flea saliva proteins in Peak N were also subjected to proteolyticcleavage in order to obtain internal amino acid sequence data.Specifically, the proteins in Peak N were cleaved with EndoproteinaseAsp-N (available from Boehringer Mannheim Biochemica, Indianapolis,Ind.) using methods standard in the art. The digested protein was thenresolved by HPLC using the method described by Stone et al. (ibid.) andsequenced as previously described. A partial amino acid sequence of fleasaliva proteins in Peak N, named fragment pfspN(100-101), was deterr,ned to be D I E N I K K G E G Q P G A P G G K E N N L S/L V L, denotedherein as SEQ ID NO: 31.

Example 5

[0290] This example describes the further characterization of proteinscontained in Peak H.

[0291] To determine the isoelectric pH of the proteins contained in PeakH, proteins present in that peak were resolved using standardisoelectric focusing techniques known to those of skill in the art; see,for example, O'Farrell, 1975, J. Biol. Chem., Vol. 250, pp. 4007-4021.The pI value for proteins contained in Peak H is about pI 9, rangingfrom about pI 8.5 to about pI 9.6.

[0292] The molecular weight of proteins contained in Peak H wasdetermined by ESMS. The ESMS procedure was performed on a Fisons VGquattro-SQ mass spectrometer. The mass range was calibrated for 100-2000m/z. The injection rate was performed at 4 μl per minute. The conevoltage was set at 45 volts. The injection sample contained 0.1% formicacid in 50% acetonitrile at a protein concentration of about 100 pmoleper μl. The results indicate that Peak H apparently contains apopulation of proteins all having a molecular weight of 8613±6 daltons.

Example 6

[0293] This example describes the isolation of nucleic acid sequencesencoding at leartL portions of flea saliva proteins fspH and fspI.

[0294] A. Description of Flea Libraries

[0295] Fed flea and unfed flea cDNA libraries were prepared usingstandard procedures. Briefly, about 3000 to 4000 fed fleas and about thesame number of unfed fleas were collected separately, placed into adry-ice cooled mortar/pestle and ground to a fine powder. RNA from theground-up fleas was prepared by direct extraction using the guanidiniumthiocyanate procedure followed by centrifugation in cesium chloridegradients (see, for example, Sambrook et al., ibid.). Cesiumchloride-purified gelatinous RNA pellets were collected and dissolved insterile TE buffer (10 mM Tris-HCl, pH 7.6 and 1 mM EDTA) containing 0.1%sodium dodecyl sulfate. The dissolved pellet was precipitated withaddition of 3 M sodium acetate, pH 5.2 to a final concentration of 0.2mM and two volumes of absolute ethanol to remove residual CsCl. TotalRNA was fractionated for enrichment of the mRNA fraction using aFastTrack™ kit (available, with procedures, from InVitrogen Corp., SanDiego, Calif.).

[0296] Isolated whole flea mRNA was used directly for cDNA synthesis andmolecular cloning. The methods of cDNA synthesis and molecular cloningwere those provided with the Lambda Zap-cDNA synthesis kit™ (availablefrom Stratagene, Inc., La Jolla, Calif.). Following is a list of fleacDNA libraries prepared having the indicated number of total plaqueforming units (PFU) packaged: (a) two whole fed flea cDNA expressionlibraries referred to as Library C (about 2.5×10⁶ PFU) and Library H(about 1.3×10⁶ PFU); (b) a whole unfed flea cDNA expression library(about 1.3×10⁶ PFU); (c) a flea salivary gland cDNA expression libraryprepared from approximately 6000 salivary glands collected from fed andunfed fleas (about 1.5×10⁶ PFU); and (d) a flea fed midgut cDNAexpression library prepared from approximately 5000 isolated midguts(about 2.3×10⁶ PFU).

[0297] B. Isolation of a Nucleic Acid Molecule Encoding fspH

[0298] A nucleic acid molecule encoding a portion of flea saliva proteinfspH was identified using the flea salivary gland cDNA expressionlibrary described in Example 6A.

[0299] Degenerate synthetic oligonucleotide primers were designed fromthe amino acid sequence deduced for fspH (see Example 4). Threesynthetic oligonucleotides were synthesized that corresponded to theregion of fspH spanning from about residues 38 through 51 of SEQ ID NO:2: Primer 1, a “sense” primer corresponding to amino acid residues fromabout 38 through about 44 of SEQ ID NO: 2, has the nucleic acid sequence5′ AAT(C) AAT(C) AAT(C) AAT(C) CCT(GAC) TGT(C) CA 3′, and is denoted SEQID NO: 15. Primer 2, an “antisense” primer corresponding to amino acidresidues from about 46 through about 51 of SEQ ID NO: 2, has the nucleicacid sequence 5′ CA C(T)TC C(TAG)CT(G) CAT G(A)CA G(A)AA 3′ and isdenoted SEQ ID NO: 16. Primer 3, a sense primer corresponding to aminoacid residues from about 43 through about 48 of SEQ ID NO: 2, has thenucleic acid sequence 5′ TGT(C) CAT(C) T(C)TG(ATC) TTT(C) TGC(T) ATG-3′and is denoted SEQ ID NO: 17. A fourth primer, Primer 4, was synthesizedthat corresponded to the carboxyl region of fspH, namely spanning fromabout amino residue 69 through 76 of SEQ ID NO: 2. Primer 4, anantisense primer, has the nucleic acid sequence 5′ GGA(CGA) GCT(C)TCA(G) CAA(G) TAA(G) CAA(G) TGT(C) TT′ 3′, denoted SEQ ID NO: 18.

[0300] PCR amplification of fragments from the flea salivary glandlibrary was conducted using standard techniques. PCR amplificationproducts were generated using the combination of Primer 1 and the M13forward universal standard primer 5′ GTAAAACGACGGCCAGT 3′, denoted SEQID NO: 19. The resultant PCR amplification products were used for anested PCR amplification using Primer 3 and Primer 4. The resultant PCRproduct, a fragment of 101 nucleotides denoted nfspH₁₀₁, was cloned intothe InVitrogen, Corp., TA™ cloning vector (procedures provided byInVitrogen, Corp.) and subjected to DNA sequence analysis using standardtechniques. The resulting nucleic acid sequence is presented as SEQ IDNO: 20: T TGT CAC TTT TTT TGT ATG AGA GAA TGC AGG TCA GGA AAC GGC GGTTGC GGA AAC GGA GGA AGG ACA AGA CCT GAT TCG AAG CAC TGC TAT GC

[0301] (primer-derived sequences are in bold). The 60 nucleotides ofinternal non-primer-derived sequence codes for 20 amino acids of fspH,spanning from residue about 48 through about 68, as numbered in SEQ IDNO: 2.

[0302] Using standard techniques, nucleic acid molecule nfspH₁₀₁ can beused as a probe to isolate a nucleic acid molecule that encodes aprotein corresponding to a full-length, or larger partial, fspH protein.

[0303] C. Isolation of a Nucleic Acid Molecule Encoding fspI

[0304] The amino acid sequence for fspI (SEQ ID NO: 6) disclosed inExample 4 was used to design a set of synthetic degenerateoligonucleotide PCR amplification primers. Degenerate Primer 5, a senseprimer corresponding to residues from about 1 through about 8 of SEQ IDNO: 6, has the nucleic acid sequence 5′ GAA(G) GAT(C) ATT(CA) TGG AAA(G)GTT(CAG) AAT(C) AA 3′, denoted SEQ ID NO: 21. Degenerate Primer 6, asense primer corresponding to residues from about 11 through about 18 ofSEQ ID NO: 6, has the nucleic acid sequence 5′ ACT(CGA) TCT(CGA)GGT(CGA) GGT(CGA) AAA(G) AAT(C) CAA(G) GA 3′, denoted SEQ ID NO: 22.

[0305] Primers 5 and 6 were used in combination with the vector primersBSKX (5′ TTGGGTACCGGGCCCCCCCT 3′, SEQ ID NO: 23) and the M13 primerdenoted by SEQ ID NO: 19 in order to generate PCR amplificationproducts. The PCR products were cloned into the InVitrogen TA™ vectorand subjected to DNA sequence analysis. One cloned product analyzed,called nfspI₅₇₃ contained a 573-nucleotide product that corresponded, atleast in part, to the partial ami~io acid sequence determined for fspI.The nucleotide sequence of nfspI₅₇₃ is presented as SEQ ID NO: 24.Translation of SEQ ID NO: 24 yields the following longest-open readingframe, denoted as SEQ ID NO: 25.

[0306] By combining the partial N-terminal sequence of fspI (SEQ ID NO:6) with the protein sequence SEQ ID NO: 25 deduced from the nucleic acidsequence SEQ ID NO: 24, it is possible to obtain an apparent full-lengthamino acid sequence for fspI, denoted SEQ ID NO: 26.

Example 7

[0307] This example further describes the isolation of nucleic acidsequences encoding at least portions of flea saliva proteins fspH andfspI.

[0308] A. Amplification of a Nucleic Acid Molecule Encoding fspH

[0309] The DNA sequence determined from the carboxyl-terminal PCRproduct (SEQ ID NO: 20) in Example 6B was used to synthesize twonon-degenerate synthetic homologous primers: Primer 7, 5′ CCT GAC CTGCAT TCT CTC ATA C 3′, denoted SEQ ID NO: 38, and Primer 8, 5′ AGG TCTTGT CCT TCC TCC GTT TCC GCA 3′, denoted SEQ ID NO: 39. Primer 8 was usedin combination with the M13 reverse primer 5′ GGAAACAGCTATGACCATG 3′,denoted SEQ ID NO: 40, to amplify the 5′-terminal portion of the fsphgene from a fraction of the salivary gland CDNA expression librarydescribed above in Example 6A using standard techniques. The resultantPCR product, although not clearly visible on a gel, was identified as asingle product by Southern hybridization using Primer 7 as a[³²P]-radiolabeled probe. A clearly visible ethidium bromide stained PCRproduct was obtained by performing a nested PCR reaction utilizingPrimer 7 and the vector T3 primer, 5′ ATTAACCCTCACTAAAG 3′, denoted SEQID NO: 41. The approximately 400-bp product was clearly visible on a 1%agarose gel and was hybridization-positive with [³²P]-labeled degeneratePrimer 1 (SEQ ID NO: 15).

[0310] A partial, 242-bp, nucleotide sequence of the 400-bp product offspH, named nfspH₂₄₂ is presented as SEQ ID NO: 32. Translation of SEQID NO: 32 yields the amino acid sequence, named PfspH₈₀ denoted as SEQID NO: 33.

[0311] B. Amplification of a Nucleic Acid Molecule Encodinq fspI

[0312] Two additional primers were made for isolating the fspI proteincDNA sequence from the flea salivary gland library prepared as describedin Example 6A. The isolation procedure used PCR reiteration of the fleasalivary gland library and probe hybridization to the PCR generatedproducts. Repeating the PCR on fractions of the flea salivary glandlibrary (mini-libraries) narrowed the occurrence frequency of the clonedfspI protein cDNA to approximately 1 in 200 plaque forming units (PFU)before a final plaque lift and identification by hybridization with a[³²P]-labeled probe.

[0313] Two primers based on SEQ ID NO: 24 were used: Primer 9, 5′ GCAAAG GTT ATA GAG GAG CTT G 3′, denoted as SEQ ID NO: 42, and Primer 10,5′ AGC TTT CCA TCA CAT CCA GC 3′, denoted as SEQ ID NO: 43. The primersgenerated an internal PCR DNA sequence of 268 bp (including primers)which was used as a marker sequence for screening the salivary glandmini-libraries. The final screening of the salivary gland mini-librarieswas done with a pool of the four [³²P]-labeled primers; Primer 5, SEQ IDNO: 21, Primer 6, SEQ ID NO: 22, Primer 8, SEQ ID NO: 42 and Primer 10,SEQ ID NO: 43, using standard techniques. A nucleic acid molecule, namednfspI₅₉₁, identified by this technique was sequenced using standardtechniques to give SEQ ID NO: 34. The translation of SEQ ID NO: 34yielded yields the amino acid sequence, denoted as SEQ ID NO: 35, for aprotein named PfspI₁₅₅. This amino acid sequence is similar to SEQ IDNO: 26, except that SEQ ID NO: 35 does not contain the amino acidsequence E D I at the amino terminus, and SEQ ID NO: 35 contains a “C”at position 7, whereas SEQ ID NO: 26 has an “L” at the correspondingposition.

Example 8

[0314] This example demonstrates the ability of a formulation of thepresent invention to induce flea allergy dermatitis in an animalsusceptible to flea allergy dermatitis.

[0315] To determine whether the isolated flea saliva proteins describedin Examples 2 and 3 were capable of inducing an allergic response inanimals susceptible to flea allergy dermatitis, skin tests wereperformed on sensitized dogs. Six dogs were sensitized to fleas usingthe method of Gross, et al., 1985, Veterinary Pathology, Vol. 22, pp.78-71. Briefly, each dog was exposed to about 25 C. felis fleascontained in chambers by allowing the contained fleas to feed on theexperimental dogs for about 15-minute periods at weekly intervals. Thesix dogs were sensitized over the following periods: Dog 2080109 wasexposed to fleas 38 times over a period spanning Aug. 31, 1993 throughJun. 7, 1994. Dog 2082101 was exposed to fleas 22 times over a periodspanning Dec. 14, 1993 through Jun. 7, 1994. Dog 2082128 was exposed tofleas 20 times over a period spanning Aug. 31, 1993 through May 24,1994. Dog BFQ2 was exposed to fleas 17 times over a period spanning Mar.15, 1994 through Jul. 12, 1994. Dog CPO2 was exposed to fleas 12 timesover a period spanning Mar. 15, 1994 through Jun. 7, 1994. Dog CQQ2 wasexposed to fleas 1 time on Mar. 15, 1994.

[0316] Skin testing was performed the morning of Jul. 21, 1994. The dogswere shaved in the lateral thorax/abdominal area and intradermallyinjected in that area with a variety of formulations of the presentinvention as well as with control solutions. The total volume perinjection was 50 microliters (μl), with the formulations and controlsbeing diluted in phenolated saline. Each dog received the injectionslisted in Table 1. TABLE 1 Samples administered to dogs. SAMPLEREPLICATES μg/inj FLEA-HOUR DILUENT 2 N/A* N/A HISTAMINE 2 1.38 N/AGREER 3 50 (w/v) N/A FS-1 3 1.88  4,660 A 3 0.23 23,000 B 3 0.32 23,000C1 3 1.10** 23,000 C2 3 0.42 23,000 D1 3 0.24 23,000 D2 3 0.29 23,000 E3 0.16 23,000 F 3 0.10 23,000 G 3 0.21 23,000 H 3 0.20 23,000 I 3 0.1223,000 J 3 0.08 23,000 K 3 0.12 23,000 L 3 0.08 23,000 M1 3 0.16 23,000M2 3 0.27 23,000 N 3 0.20 23,000 FS-2 3 0.71  4,660

[0317] Note that in these studies, fspJ1 and fspJ2 were administeredtogether as fspJ; fspL1 and fspL2 were administered together as fspL;fspN1, fspN2 and fspN3 were administered together as fspN. It is also tobe noted that A, B, C1, C2, D1, D2, E, F, G, H, I, J, K, L, M1, M2 and Nrefer, respectively to flea saliva proteins fspA, fspB, fspC1, fspC2,fspD1, fspD2, fspE, fspF, fspG, fspH, fspI, fspJ, fspK, fspL, fspM1,fspM2 and fspN. The negative control comprised diluent (NC) and thepositive controls comprised Greer antigen (GR) and histamine (HIS). Theamount of Greer antigen used was determined by weight per volume (w/v)according to the information provided by the manufacturers (GreerLaboratories, Inc., Lenoir, N.C.). The amount of histamine used wasdetermined by information provided on the supplier's label (availablefrom Greer Laboratories, Inc., Lenoir, N.C.).

[0318] A. Comparison of Wheal Sizes at Sites of Injection

[0319] All injection sites were objectively (Obj) measured inmillimeters (mm) at 15 min and subjectively (Sub) scored on a scale of 0to 4. The subjective scoring was performed by Kenneth W. Kwochka,D.V.M., Diplomat ACVD, (American College of Veterinary Dermatologists)at Ohio State University, Columbus, Ohio. Tables 2 through 7 indicatethe results obtained for each dog. # refers to the number designationgiven to each sample; antigen refers to the sample. Inj 1, Inj 2 and Inj3 refer to triplicate injections and NA refers to “not applicable.”TABLE 2 DOG ID: 2082101 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 # AntigenSub Obj Sub Obj Sub Obj 1 Neg Cntl 0 6 NA NA NA NA 2 Histamine 4 12 NANA NA NA 3 Greer 3 10 3 10 3 10 4 FS-1 3 10 4 12 4 12 5 A 1 8 0 8 0 8 6B 0 6 0 6 0 6 7 C1 0 6 0 6 0 6 8 C2 0 6 0 6 0 6 9 D1 0 8 0 8 0 6 10 D2 06 0 6 0 8 11 E 3 12 3 12 3 12 12 F 3 14 3 12 3 12 13 G 3 12 3 12 3 12 14H 3 11 2 12 3 12 15 I 3 12 2 12 3 11 16 J 2 10 2 11 2 10 17 K 2 11 2 102 9 18 L 2 9 1 10 1 10 19 M1 2 12 2 11 2 11 20 M2 3 12 3 11 3 12 21 N 311 3 10 2 11 22 FS-2 2 11 3 12 2 10 23 Neg Cntl 0 8 NA NA NA NA 24Histamine 4 14 NA NA NA NA

[0320] TABLE 3 DOG ID: 2080109 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 #Antigen Sub Obj Sub Obj Sub Obj 1 Neg Cntl 0 7 NA NA NA NA 2 Histamine 414 NA NA NA NA 3 Greer 0 8 0 8 0 8 4 FS-1 4 13 4 13 4 13 5 A 0 9 0 8 0 86 B 0 7 0 7 0 7 7 C1 0 8 0 7 0 7 8 C2 0 8 0 7 0 8 9 D1 1 9 1 9 1 9 10 D21 9 1 8 1 8 11 E 3 11 3 11 2 10 12 F 3 11 3 13 4 13 13 G 3 14 3 13 3 1314 H 2 12 2 11 2 10 15 I 2 10 3 10 3 10 16 J 2 10 3 10 3 10 17 K 2 9 2 92 9 18 L 1 9 1 6 1 7 19 M1 3 11 3 13 3 13 20 M2 3 14 3 13 3 14 21 N 3 133 14 2 10 22 FS-2 2 9 1 7 1 8 23 Neg Cntl 0 6 NA NA NA NA 24 Histamine 416 NA NA NA NA

[0321] TABLE 4 DOG ID: 2082128 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 #Antigen Sub Obj Sub Obj Sub Obj 1 Neg Cntl 0 6 NA NA NA NA 2 Histamine 412 NA NA NA NA 3 Greer 0 6 0 6 0 6 4 FS-1 3 12 3 12 3 12 5 A 0 7 0 7 0 66 B 0 7 0 7 0 6 7 C1 0 7 0 6 0 7 8 C2 0 6 0 7 0 7 9 D1 0 7 0 7 0 7 10 D20 7 0 7 0 7 11 E 0 7 0 6 0 7 12 F 0 6 0 6 0 6 13 G 1 10 1 9 1 9 14 H 210 2 10 2 11 15 I 3 12 3 12 3 11 16 J 3 12 3 11 3 11 17 K 3 11 3 12 3 1218 L 3 11 3 10 3 11 19 M1 3 11 3 11 3 12 20 M2 3 12 3 12 3 12 21 N 3 123 12 3 12 22 FS-2 3 12 3 11 3 12 23 Neg Cntl 0 6 NA NA NA NA 24Histamine 4 14 NA NA NA NA

[0322] TABLE 5 DOG ID: BFQ2 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 #Antigen Sub Obj Sub Obj Sub Obj 1 Neg Cntl 0 6 NA NA NA NA 2 Histamine 412 NA NA NA NA 3 Greer 0 6 0 6 0 6 4 FS-1 1 9 1 9 1 9 5 A 0 7 0 7 0 7 6B 0 7 0 7 0 7 7 C1 0 7 1 7 1 7 8 C2 0 7 0 7 0 6 9 D1 0 8 1 7 1 8 10 D2 07 0 6 1 7 11 E 1 7 0 6 0 6 12 F 1 6 1 7 0 7 13 G 0 8 1 8 1 8 14 H 0 8 07 0 7 15 I 1 7 0 7 0 8 16 J 0 7 0 7 0 7 17 K 0 7 0 7 0 6 18 L 0 8 0 7 07 19 M1 0 7 0 7 0 7 20 M2 0 7 0 7 1 8 21 N 3 12 3 11 3 11 22 FS-2 3 11 311 3 11 23 Neg Cntl 0 7 NA NA NA NA 24 Histamine 4 15 NA NA NA NA

[0323] TABLE 6 DOG ID: CPO2 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 #Antigen Sub Obj Sub Obj Sub Obj 1 Neg Cntl 0 3 NA NA NA NA 2 Histamine 413 NA NA NA NA 3 Greer 0 7 0 7 0 6 4 FS-1 4 12 4 12 4 12 5 A 0 7 0 6 0 66 B 0 6 0 7 0 7 7 C1 0 7 0 6 0 7 8 C2 0 6 0 6 0 6 9 D1 0 7 1 7 0 7 10 D21 6 0 6 0 5 11 E 0 6 0 6 0 6 12 F 0 6 0 6 2 7 13 G 2 9 2 8 2 8 14 H 4 114 12 4 11 15 I 3 12 3 11 3 10 16 J 3 10 3 11 3 10 17 K 2 8 2 8 2 8 18 L1 8 1 7 1 7 19 M1 3 11 3 11 3 11 20 M2 3 11 4 12 4 12 21 N 4 12 3 10 311 22 FS-2 3 11 3 12 3 12 23 Neg Cntl 0 6 NA NA NA NA 24 Histamine 4 13NA NA NA NA

[0324] TABLE 7 DOG ID: CQQ2 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 #Antigen Sub Obj Sub Obj Sub Obj 1 Neg Cntl 0 6 NA NA NA NA 2 Histamine 413 NA NA NA NA 3 Greer 0 7 0 7 0 7 4 FS-1 2 8 2 8 2 8 5 A 0 6 0 6 0 7 6B 0 7 0 7 0 6 7 C1 0 7 0 6 0 6 8 C2 0 7 0 7 0 6 9 D1 0 6 0 6 0 6 10 D2 06 0 6 0 7 11 E 0 6 0 6 0 6 12 F 0 6 0 7 0 7 13 G 0 7 0 7 0 6 14 H 1 7 17 1 7 15 I 2 8 2 9 2 8 16 J 2 8 2 8 2 8 17 K 1 7 1 7 1 7 18 L 1 6 0 6 06 19 M1 2 7 2 8 2 8 20 M2 2 8 2 8 2 9 21 N 3 11 3 12 3 11 22 FS-2 3 11 311 3 10 23 Neg Cntl 0 7 NA NA NA NA 24 Histamine 4 14 NA NA NA NA

[0325] As a control, 2 flea naive dogs (i.e., dogs that had never beenexposed to fleas) were also tested with single replicates of the samesamples that were injected into the sensitized dogs. These dogs arereferred to as WANU and WBCE. Objective and subjective wheal sizemeasurements 15 minutes after injection of the samples are shown inTables 8 and 9. TABLE 8 DOG ID: WANU Inj 1 Inj 1 # Antigen Sub Obj 1 NegCntl 0 7 2 Histamine 4 10 3 Greer 0 6 4 FS-1 0 6 5 A 0 7 6 B 0 6 7 C1 06 8 C2 0 6 9 D1 0 7 10 D2 0 6 11 E 0 6 12 F 0 6 13 G 0 7 14 H 0 7 15 I 07 16 J 0 7 17 K 0 6 18 L 0 7 19 M1 0 6 20 M2 0 6 21 N 1 8 22 FS-2 1 8 23Neg Cntl NA NA 24 Histamine NA NA

[0326] TABLE 9 DOG ID: WBCE Inj 1 Inj 1 # Antigen Sub Obj 1 Neg Cntl 0 62 Histamine 4 12 3 Greer 0 7 4 FS-1 0 7 5 A 0 7 6 B 0 7 7 C1 0 7 8 C2 07 9 D1 0 7 10 D2 0 6 11 E 0 7 12 F 0 7 13 G 0 8 14 H 0 7 15 I 0 7 16 J 07 17 K 0 7 18 L 0 6 19 M1 0 7 20 M2 0 7 21 N 0 7 22 FS-2 0 7 23 Neg CntlNA NA 24 Histamine NA NA

[0327] The average subjective score obtained for each flea salivaantigen from the 6 sensitized dogs tested was calculated and issummarized in FIG. 5. The results indicate that the flea saliva productsproduced as described in Examples 2 and 3 include at least oneallergenic protein capable of inducing an immediate hypersensitiveresponse in a sensitized dog. In particular, injection of the mixturesof flea saliva antigens referred to as FS-1 and FS-2 resulted insubstantial wheal formation. Flea saliva proteins fspE, fspF, fspG,fspH, fspI, fspJ, fspK, fspL, fspM1, fspM2 and fspN also resulted insubstantial wheal formation. Flea saliva proteins fspA, fspB, fspC1,fspC2, fspD1 and fspD2 produced minimal, if any, allergic response,depending on the dog being tested. The sample containing fspH producedthe largest wheal formation when compared with the other flea salivaproteins.

[0328] *B. Comparison of Levels of Induration and Erythema at theInjection Sites

[0329] In addition to wheal size, the amount of induration and erythemawere also measured at each site of injection. Induration produced by theinjection of the flea saliva antigens was measured at 6 hours and 24hours by subjective scoring. Such subjective induration measurementswere performed by Kenneth W. Kwochka, D.V.M. In addition, the amount oferythema at each site of injection were subjectively scored by KennethW. Kwochka, D.V.M.

[0330] The amounts of induration and erythema measured by subjectivescoring at 6 hours were negative for each of the sensitized and controldogs except for the following formulations in the following sensitizeddogs. Administration of FS-1 to Dog 2082101 produced an averageinduration score of 1 at 2 sites of injection but no erythema score.Administration of fspL to Dog 2082101 produced no induration but anerythema score of 1 at 1 site of injection. Administration of fspM1 toDog 2082101 produced no induration but an erythema score of 3 at 1 siteof injection. Administration of FS-2 to Dog 2082101 produced noinduration but an average erythema score of 1.33 at 3 sites ofinjection. Administration of fspH to Dog 2082128 produced no indurationbut an average erythema score of 2 at 3 sites of injection.Administration of fspI to Dog 2082128 produced an average indurationscore of 1 and an average erythema score of 1 at 2 sites of injection.Administration of fspJ to Dog 2082128 produced an average indurationscore of 1 and an average erythema score of 1 at 3 sites of injection.Administration of FS-2 to Dog 2082128 produced no induration but anaverage erythema score of 2 at 3 sites of injection.

[0331] Administration of FS-1 to Dog BFQ2 produced an average indurationscore of 2 and an average erythema score of 2 at 3 sites of injection.Administration of fspN to Dog BFQ2 produced an average induration scoreof 1 and an average erythema score of 2 at 2 sites of injection.Administration of FS-2 to Dog BFQ2 produced an average induration scoreof 1 and an average erythema score of 2 at 2 sites of injection.

[0332] Administration of FS-1 to Dog CPO2 produced an average indurationscore of 2.5 but no erythema at 2 sites of injection. Administration offspG to Dog CPO2 produced no induration but an average erythema score of2 at 3 sites of injection. Administration of fspH to Dog CPO2 producedno induration but an average erythema score of 1 at 2 sites ofinjection. Administration of FS-2 to Dog CPO2 produced no induration butan average erythema score of 2 at 3 sites of injection.

[0333] The average subjective score for induration obtained for eachflea saliva antigen from the 6 sensitized dogs tested was calculated andis summarized in FIG. 6. The average subjective score for erythemaobtained for each flea saliva antigen from the 6 sensitized dogs testedwas calculated and is summarized in FIG. 7.

[0334] The amounts of induration and erythema measured by subjectivescoring at 24 hours results for five of the flea-sensitized dogs and thetwo control dogs were negative except for the following formulations inthe following sensitized dogs.

[0335] Administration of fspI to Dog 2082101 produced an averageinduration score of 1 and an average erythema score of 1 at 3 sites ofinjection. Administration of fspJ to Dog 2082101 produced an averageinduration score of 1 and an average erythema score of 1 at 3 sites ofinjection. Administration of fspM1 to Dog 2082101 produced an averageinduration score of 1 and an average erythema score of 3 at 3 sites ofinjection. Administration of fspN to Dog 2082101 produced an averageinduration score of 1 and an average erythema score of 2 at 3 sites ofinjection. Administration of FS-2 to Dog 2082101 produced an averageinduration score of 3 and an average erythema score of 4 at 3 sites ofinjection.

[0336] Administration of FS-1 to Dog BFQ2 produced an average indurationscore of 1 and an average erythema score of 1 at 3 sites of injection.Administration of FS-2 to Dog BFQ2 produced an induration score of 1 andan erythema score of 1 at 1 site of injection.

[0337] Administration of FS-1 to Dog CPO2 produced an induration scoreof 2 and an erythema score of 1 at 1 site of injection. Administrationof fspI to Dog CPO2 produced an average induration score of 1 and anaverage erythema score of 1 at 3 sites of injection. Administration ofFS-2 to Dog CPO2 produced an average induration score of 1 and anaverage erythema score of 2 at 3 sites of injection.

[0338] Administration of Greer antigen to Dog CQQ2 produced noinduration but an average erythema score of 1 at 3 sites of injection.Administration of FS-1 to Dog CQQ2 produced an induration score of 1 andan erythema score of 1 at 1 site of injection. Administration of fspI,fspJ, fspM1 or fspM2 to Dog CQQ2 produced no induration but an averageerythema score of 1 at 3 sites of injection. Administration of fspN toDog CQQ2 produced an induration score of 1 and an erythema score of 1 at1 site of injection. Administration of FS-2 to Dog CQQ2 produced anaverage induration score of 1 and an average erythema score of 2 at 3sites of injection.

[0339] The average subjective score for induration obtained for eachflea saliva antigen from the 6 sensitized dogs tested was calculated andis summarized in FIG. 8. The average subjective score for erythemaobtained for each flea saliva antigen from the 6 sensitized dogs testedwas calculated and is summarized in FIG. 9.

[0340] The results indicate that at least some of the flea salivaprotein formulations produced as described in Examples 2 and 3 includeat least one allergenic protein capable of inducing a delayedhypersensitive response in a sensitized dog. Injection of the mixturesof flea saliva proteins referred to as FS-1 and FS-2 induced substantialinduration and erythema for at least 24 hours. In addition, the fleasaliva protein samples fspI, fspJ, M1 and fspN were sufficientlyallergenic to induce induration and erythema for at least 24 hours. Theflea saliva protein sample fspL and fspM2 induced substantial levels ofinduration but not substantial levels of erythema at 24 hours.

[0341] Taken together, the results shown indicated above and shown inFIGS. 5 through 9, indicate that saliva protein formulations of thepresent invention are sufficiently allergenic to induce a hypersensitiveresponse in a sensitized dog. Numerous samples induced both an immediatehypersensitive response and a delayed hypersensitive response.

Example 9

[0342] This example demonstrates the ability of numerous flea salivaprotein samples isolated in Examples 2 and 3 to induce a hypersensitiveresponse by histopathology of tissue removed from selected lesions onthe dogs described in Example 8.

[0343] Two tissue samples per dog were removed from each sensitized dogdescribed in Example 8. No biopsies were taken from the two naive dogs.The selected sites from which the tissue samples were removed areindicated in Table 10 below. Biopsies were taken with a 4 mm biopsypunch after subcutaneous injections of Lidocaine. Biopsies wereprocessed and read by Dr. David M. Getzy, DVM, Diplomat ACVP (AmericanCollege of Veterinary Pathologists) at the Colorado VeterinaryDiagnostic Laboratory (College of Veterinary Medicine and BiomedicalSciences, Colorado State University, Fort Collins, Colo.). TABLE 10Histopathology Dog Antigen Time No. Slide Lesion Type Grade 101 FS-1 15min. 1 A A 1  6 hr. 2 B A 2.5 24 hr. 3 C A 3 109 FS-1 15 min. 4 D A 1  6hr. 5 E C 2 24 hr. 6 F C 3 128 FS-1 15 min. 7 G A 1.5  6 hr. 8 H C 1.524 hr. 9 I C 3 CPO2 FS-1 15 min. 10 J A 1.5  6 hr. 11 K C 3 24 hr. 12 LC 4 CQQ2 FS-1 15 min. 13 M A 1.5  6 hr. 14 N C 2.5 24 hr. 15 O C 2.5 101fspE 15 min. 16 P A 1  6 hr. 17 Q C 1.5 24 hr. 18 R A 1.5 109 fspF 15min. 19 S A 1  6 hr. 20 T A 1.5 24 hr. 21 U A 1.5 128 fspI 15 min. 22 VA 1  6 hr. 23 W C 2.5 24 hr. 24 X C 2.5 BFQ2 fspN 15 min. 25 Y A 1.5  6hr. 26 Z C 2 24 hr. 27 AA C 3.5 BFQ2 fspO 15 min. 28 BB A 1  6 hr. 29 CCC 3 24 hr. 30 DD C 2.5 CPO2 fspH 15 min. 31 EE A 1.5  6 hr. 32 FF C 1.524 hr. 33 GG A 1.5 CQQ2 fspN 15 min. 34 HH A 1  6 hr. 35 II C 2.5 24 hr.36 JJ C 2.5

[0344] Two types of lesions were found in the tissue samples tested.Lesion Type A refers to a moderate superficial dermal edema having mildnumbers of mast cells in a perivascular orientation within thesuperficial dermis. Vascular endothelium exhibited mild reactivehypertrophy. Minimal numbers of neutrophils were noted in this region aswell. Lesion Type C refers to lesions that were similar to thosedescribed in Lesion Type A except that the eosinophils were mild tomoderate in severity, while neutrophils and mast cells were mild inseverity.

[0345] On a scale of 0 to 5, lesions ranged from grade 1 to grade 4 inseverity. Some of the specimens had predominantly mastocyticinflammatory perivascular infiltrates, edema, and minimal numbers ofother cellular components. Other sections showed a predominance ofeosinophilic inflammatory infiltrates, with lesser numbers of mast cellsand neutrophils. The severity of these lesions was variable, however, insome areas, it progressed to intraepidermal eosinophilic pustulation andcollagen necrobiosis within the superficial dermis.

[0346] Taken together, the tissue samples indicated the presence ofsuperficial perivascular/periadnexal, mastocytic and eosinophilic,subacute dermatitis. Lesions noted in all the slide specimens examinedare consistent with an allergic Type I hypersensitivity reaction.

Example 10

[0347] This example further demonstrates the ability of proteinsdescribed in Examples 2 and 3 to induce an allergic response in animalsnaturally susceptible to flea allergy dermatitis through skin testsperformed on dogs. These reactions were compared to those obtained usingthe current standard for diagnosis of flea allergy dermatitis, GreerWhole Flea Extract (Greer Laboratories, Inc., Lenoir, N.C.). Inaddition, in order to determine specificity of the reactions, testresults were compared to those obtained from a population of controldogs with normal skin and a population of dogs with pruritic skindisorders other than flea allergy dermatitis.

[0348] Three groups of dogs were used in the study: (1) 10 dogs withnaturally occurring flea allergy dermatitis as determined by clinicalsigns, presence of fleas at the time of diagnosis, and a positiveimmediate or delayed reaction to Greer Whole Flea Extract; (2) 10 dogswith non-flea-related pruritic dermatoses including, but not limited to,atopy, food allergy dermatitis, pyoderma, seborrhea, and other parasitichypersensitivity reactions; and (3) 10 dogs with normal skin and nohistory of chronic skin diseases. The dogs were of any breed, age orsex. They were recruited from the hospital population of the Ohio StateUniversity Veterinary Teaching Hospital, Columbus, Ohio. All dogs hadwritten owner consent to participate in the study.

[0349] All tests and subjective scoring were performed by Kenneth W.Kwochka, D.V.M., Diplomat ACVD, (American College of VeterinaryDermatologists), in the Dermatology Examination Room at the VeterinaryTeaching Hospital, College of Veterinary Medicine, The Ohio StateUniversity, Columbus, Ohio. All dogs were tested on theanterior-ventral-lateral aspect of the chest on the left side. Dogs weresedated for t sting using standard dosages of xylazine and atropineadministered intravenously immediately before the skin test. Noglucocorticoids, antihistamines, or other non-steroidal antiinflamatorymedications were allowed for at least 3 weeks prior to testing. The areafor testing was gently clipped with a #40 electric clipper blade and theinjection sites marked with an indelible black felt-tipped marking pen.Twenty-two sites were marked: two rows of ten dots and one row of twodots. Intradermal injections were placed both above and below each markfor a total of forty-four injections that were administered in thefollowing order:

[0350] Row 1: Neg. cont.-Histamine-Greer-Greer-Flea saliva-Fleasaliva-A-A-B-B

[0351] Row 2: C1-C1-C2-C2-D1-D1-D2-D2-E-E

[0352] Row 3: F-F-G-G-H-H-I-I-J-J-

[0353] Row 4: K-K-L-L-M1-M1-M2-M2-N-N

[0354] Row 5: FS2-FS2

[0355] Row 6: Neg. cont.-Histamine

[0356] Each site was injected intradermally with 50 μl of sterilediluent (Neg. cont.), 1/100,000 w/v histamine phosphate (Histamine),Greer Whole Flea Extract (Greer), whole flea saliva (Flea saliva), orindividual salivary protein fractions (fspA,(A); fspB, (B); fspC1, (C1);fspC2, (C2); fspD1, (D1); fspD2, (D2); fspE, (E); fspF, (F); fspG, (G);fspH, (H); fspI, (I); fspJ, (J); fspK, (K); fspL, (L); fspM1, (M1);fspM2, (M2); fspN, (N); and FS-2 (FS2). All injections were diluted inthe same sterile diluent as the Neg. cont.

[0357] Skin reactions were read subjectively and objectively at 15minutes and 24 hours after injections. Owners were required to returntheir dogs to the Veterinary Teaching Hospital for the 24 hour readings.Subjective assessments were basted on a scale of 0, 1+, 2+, 3+ and 4+based on wheal size, amount of erythema and amount of induration.Objective assessment was based on wheal diameter measured inmillimeters.

[0358] Comparison of Skin Reactions:

[0359] A. FAD Dogs:

[0360] Of the 10 dogs positive to Greer, 7 (70%) were positive the FleaSaliva (FS). None of the 3 FS-negative dogs reacted to any of thesalivary protein fractions. Additionally, the 3 dogs negative to FS at15 minutes were negative to everything at 24 hours. The 7 FS-positivedogs were used to summarize the 15 minute reactions, shown below inTable 11. TABLE 11 Immediate (15 min) subjective scores of 7 FS-positivedogs to test antigens % Positive Scores ≧2+ Scores ≧3+ 0 I 14 B, I, J, LB, D1, J, L 29 A, C1, C2, D1 A, C1, C2, K 43 E, F, K D2, E, F, H, M2 57D2, H, M2 G, N, FS2 71 G, M1 M1 86 N, FS2 Greer 100 Greer, FS FS

[0361] Four of the 7 FS-positive dogs could not be evaluated at 24 hoursbecause the severity of the immediate reactions warrantedantiinflammatory therapy. The remaining 3 FS-positive dogs were used tosummarize the 24 hour reactions, shown below in Table 12. TABLE 12Delayed (24 hr) subjective scores of 3 FS-positive dogs to test antigens% Positive Scores ≧2+ Scores ≧3+ 0 33 M2 67 Greer, FS, N, FS, N, FS2 FS2100

[0362] B. Normal Dogs:

[0363] Three dogs had an immediate reaction to the skin test antigens tosome extent. None had a positive delayed reaction at 24 hours. A summaryof the immediate (15 min) subjective results is shown below in Table 13.TABLE 13 Immediate (15 min) subjective scores of 10 normal dogs to testantigens % Positive Scores ≧2+ Scores ≧3+ 0 10 N, FS2 FS2 20 Greer, FSGreer, FS 30 40 50 60 70 80 90 100

[0364] Individual Dog Comments:

[0365] Dog #1: Greer 3+, FS 3+, N 2+, FS2 4+

[0366] Dog #2: Greer 3+

[0367] Dog #3: FS 3+

[0368] C. Non-FAD Pruritis Dogs:

[0369] Six dogs had an immediate reaction to the skin test antigens tosome extent. A summary of the immediate (15 min) subjective results isshown below in Table 14. TABLE 14 Immediate (15 min) subjective scoresof 10 Non-FAD pruritis dogs to test antigens % Positive Scores ≧2+Scores ≧3+ 0 10 G, O G, O 20 Greer, M1 Greer, FS, M1, M2 30 FS, M2, N N40 50 60 70 80 90 100

[0370] Individual Dog Comments:

[0371] Dog #1: FS 2+, M1 3+, M2 3+, N 3+, FS2 3+

[0372] Atopic dog under chronic flea exposure

[0373] Dog #2: FS 4+, G 4+, M1 4+, M2 3+, N 3+

[0374] Atopic dog under chronic flea exposure

[0375] Dog #3: FS 4+, M2 2+

[0376] Atopic dog under chronic flea exposure

[0377] Dog #4: N 3+

[0378] Atopic dog under chronic flea exposure

[0379] Dog #5: Greer 4+

[0380] Chronic otitis externa

[0381] Dog #6: Greer 4+

[0382] Generalized demodicosis (mange)

[0383] Dogs #1, #2 and #3 all came back to the clinic subsequently andwere diagnosed with FAD and were Greer positive.

[0384] Three dogs had a delayed reaction to the skin test antigens tosome extent. A summary of the delayed (24 hr) subjective results isshown below in Table 15. TABLE 15 Delayed (24 hr) subjective scores of10 Non-FAD pruritis dogs to test antigens % Positive Scores ≧2+ Scores≧3+ 0 10 FS, N, FS2 Greer, FS, N, FS2 20 30 Greer 40 50 60 70 80 90 100

[0385] Individual Dog Comments:

[0386] Dog #3: Greer 2+

[0387] Atopic dog under chronic flea exposure

[0388] Dog #4: Greer 2+

[0389] Atopic dog under chronic flea exposure

[0390] Dog #6: Greer 3+, FS 3+, N 3+, FS2 3+

[0391] Generalized demodicosis (mange)

[0392] As an aid in determining the fraction(s) of flea saliva thatcorrelate best with a positive skin test result, all the data for theartificially sensitized and clinically diagnosed FAD dogs that were 2+or greater to FS (12 dogs total; 5 artificially sensitized and 7clinically diagnosed as FAD positive) were tabulated according to theresponses to the test antigens. The immediate (15 min) subjectiveresults are shown below in Table 16, and the delayed (24 h) subjectiveresults are shown below in Table 17. TABLE 16 PERCENT RESPONDING (15 minsubjective score) Artificially Clinical Sensitized (5) Diagnosis (7)Combined (12) Score Score Score Score Score Score Antigen ≧2+ ≧3+ ≧2+≧3+ ≧2+ ≧3+ Greer 20 20 100 86 67 58 FS 100 80 100 100 100 92 A 0 0 2929 17 17 B 0 0 14 14 8 8 C 0 0 29 29 17 17 D1 0 0 29 14 17 8 D2 0 0 5743 33 25 E 40 20 43 43 42 33 F 40 40 43 43 42 42 G 60 40 71 57 67 50 H80 20 57 43 67 33 I 100 40 14 0 50 17 J 100 40 14 14 50 25 K 80 20 43 2958 25 L 20 20 14 14 17 17 M1 100 60 71 71 83 67 M2 100 80 57 43 75 58 N100 60 86 57 92 58 FS2 80 60 86 57 83 58

[0393] TABLE 17 PERCENT RESPONDING (24 hr subjective score) ArtificiallyClinical Sensitized (5) Diagnosis (7) Combined (12) Score Score ScoreScore Score Score Antigen ≧2+ ≧3+ ≧2+ ≧3+ ≧2+ ≧3+ Greer 0 0 67 0 25 0 FS0 0 67 67 25 25 A 0 0 0 0 0 0 B 0 0 0 0 0 0 C 0 0 0 0 0 0 D1 0 0 0 0 0 0D2 0 0 0 0 0 0 E 0 0 0 0 0 0 F 0 0 0 0 0 0 G 0 0 0 0 0 0 H 0 0 0 0 0 0 I0 0 0 0 0 0 J 0 0 0 0 0 0 K 0 0 0 0 0 0 L 0 0 0 0 0 0 M1 20 0 0 0 13 0M2 0 0 33 0 13 0 N 20 0 67 67 38 25 FS2 60 20 67 67 63 38

[0394] The results of these studies indicate that the most substantialresponses were obtained for fractions fspG, fspH, fspM1, fspM2 and fspN.

Example 11

[0395] The following example illustrates the expression of fspI proteinsin bacteria and in insect cells.

[0396] A. Expression of Flea Protein fspI in E. coli.

[0397] A 500 bp DNA fragment of fspI was PCR amplified from nucleic acidmolecule nfspI₅₉₁, using: Primer 11, a sense primer having the nucleicacid sequence 5′ ATTCGGATCCATGGAAAGTTAATAAAAAATGTAC 3′ (BamHI site inbold), denoted as SEQ ID NO: 36; and Primer 12, an antisense primerhaving the nucleic acid sequence 5′ TAATGGATCCTTATTTTTTGGTCGACAATAAC 3′,denoted SEQ ID NO: 37. The PCR product, a fragment of about 535nucleotides, denoted nfspI₅₃₅, was digested with BamHI restrictionendonuclease, gel purified, and subcloned into expression vectorpTrcHisB (available from InVitrogen Corp.) that had been digested withBamHI and CIP treated to produce recombinant molecule pHis-nfspI₅₃₅.

[0398] The recombinant molecule was transformed into both HB101(available from BRL, Gaithersburg, Md.) and BL21 (available fromNovagen, Madison, Wis.) competent cells to form recombinant cellsE.coliHB:pHis-nfspI₅₃₅ and E.coliBL:pHis-nfspI₅₃₅. The recombinant cellswere cultured in an enriched bacterial growth medium containing 0.1mg/ml ampicillin and 0.1% glucose at 32° C. When the cells reached anOD₆₀₀ of about 0.4-0.5, expression was induced by the addition of 0.5 mMisopropyl B-D-thiogalactoside (IPTG), and the cells were cultured for 2hours at 32° C.

[0399] SDS-polyacrylamide gel electrophoresis and western immunoblotanalyses of recombinant cell lysates containing the fusion proteinPHIS-PfspI₁₅₅ were accomplished by standard procedures using either a T7Tag monoclonal antibody (available from InVitrogen Corp.) or rabbitanti-FAD antiserum (#A3381) generated by Paravax, Inc. in Fort Collins,Colo., produced by immunizing rabbits with nitrocellulose membranescontaining flea saliva, produced as described in Example 2.Antigen/antibody reactions were detected by colorimetric enzymereactions using alkaline phosphatase-conjugated anti-mouse oranti-rabbit antibodies. A 28 kD protein was detected on the immunoblotsof induced lysates with both primary antibodies.

[0400] B. Expression of Flea Protein fspI in Insect Cells

[0401] Nucleic acid molecule nfspI₄₇₅ was PCR amplified from an fspInucleic acid molecule using the following primers, which were designedto facilitate expression in insect cells using a baculovirus vector:sense Primer 13, with the BamHI site in bold, is 5′ CGC GGA TCC TAT AAATAT GGA GGA CAT CTG GAA AGT TAA TAA AAA ATG TAC ATC 3′, denoted as SEQID NO: 44; and antisense Primer 14, with the XbaI site in bold, is 5′GCT CTA GAG CAT TTA TTT TTT GGT CGA CAA TAA CAA AAC 3′, denoted as SEQID NO: 45. The PCR product was digested with BamHI and XbaI and an about475 bp DNA fragment was excised and purified from an agarose gel.

[0402] Nucleic acid molecule nfspI₄₇₅ was ligated into a pVL1393 vector(available from InVitrogen Corp.) digested with BamHI and XbaI toproduce recombinant molecule pVL-nfspI₄₇₅.

[0403] The recombinant molecule was transfected into S. frugiperda Sf9cells with linearized Baculovirus DNA to form recombinant cell S.frugiperda:pVL-nfspI₄₇₅. The recombinant cells were cultured usingstandard conditions to produce recombinant virus. The transfectionsupernatant was also found to contain a 23 kD protein which reacted witha rabbit anti-FAD antiserum (#A3381) by Western blot analysis.

Example 12

[0404] This example describes the isolation of nucleic acid sequencesencoding at least portions of flea saliva proteins in fspN, and theircharacterization relative to human prostatic acid phosphatase.

[0405] The flea salivary gland and the whole fed flea cDNA librariesdescribed previously in Example 6A were immunoscreened using New ZealandWhite rabbit antiserum developed against a collected mixture of fleasalivary proteins (e.g., the rabbit was immunized one or more times witha ground up nitrocellulose filter used as collection membrane to collectflea saliva proteins, followed by one or more immunization with a fleasaliva protein extract eluted from a Duropore filter). Theimmunoscreening protocols used are those described in the picoBlue™Immunoscreening Kit instruction manual, available from Stratagene, Inc.The methods for preparation of the cDNA expression libraries forimmunoscreening, i.e., expression of the cDNA clones and procedures fortransferring lambda phage plaques to membranes for immunoscreening, aredescribed in the ZAP-cDNA Synthesis Kit instruction manual, alsoavailable from Stratagene, Inc., La Jolla, Calif.

[0406] Forty immunopositive clones were selected from the screening. Oneimmunopositive clone was derived from the salivary gland cDNA libraryand 39 other immunopositive clones were derived from the whole fed fleacDNA library. The initial fspN-protein cDNA sequences, termed nfspN(A)and nfspN(B) were isolated from the whole fed flea cDNA library and camefrom this initial immunoscreening.

[0407] Partial nucleotide sequences for nfspN(A) and nfspN(B) arerepresented by SEQ ID NO's. Each sequence represents approximately thecarboxyl terminal half of the cDNA gene coding region as well as the 3′untranslated region through the poly (A) region. The nucleotide sequencefor a nfspN(A) nucleic acid molecule named nfspN(A)₆₄₆ is denoted as SEQID NO: 50. Translation of SEQ ID NO: 50 yields a protein namedPfspN(A)₁₇₂ having an amino acid sequence denoted SEQ ID NO: 51. Thenucleotide sequences for a nfspN(B) nucleic acid molecule namednfspN(B)₆₁₂ is denoted SEQ ID NO: 52. Translation of SEQ ID NO: 52yields a protein named PfspN(B)₁₅₃ having an amino acid sequence denotedSEQ ID NO: 53.

[0408] In addition, an apparent N-terminal amino acid sequence deducedfrom nucleic acid sequence of nfspN(A), named PfspN(A)₅₆ and denoted SEQID NO: 54, was determined. The amino acid sequence of PfspN(A)₅₆ (i.e.,SEQ ID NO: 54) is similar, but not identical, to the N-terminal aminoacid sequences obtained for fspN1(SEQ ID NO: 11), fspN2 (SEQ ID NO: 12)and fspN3 (SEQ ID NO: 13). While not being bound by theory, it isbelieved that there is a family of fspN proteins that are found in fleasaliva, which may be due to allelic variation or multiple genes in theflea genome. Nucleic acid molecules nfspN(A)₆₄₆ and nfspN(B)₆₁₂ areabout 76% identical, and the translated products are about 65%identical.

[0409] In a second immunoscreening experiment in which antiserumcollected from a rabbit that was immunized with the proteins in peak Nof the HPLC separation of flea saliva extract described in Example 4(i.e., fspN proteins) was used to probe a flea salivary gland cDNAlibrary (prepared as described in Example 6), approximately 20 positiveclones were isolated. The nucleic acid sequence of one of the recoverednucleic acid molecules appears to be identical to that of nfspN(A). Atleast two of the other nucleic acid molecules have nucleic acidsequences that are similar, but not identical, to that of nfspN(A),again supporting the likelihood of a family of fspN proteins in fleasaliva. Yet another nucleic acid molecule appears to have a nucleic acidsequence that is similar to myosin gene sequences.

[0410] The nucleic acid and amino acid sequences of the fspN(A) andfspN(B) nucleic acid molecules and proteins, respectively, were comparedto known nucleic acid and amino acid sequences using a Genbank homologysearch. Both nucleic acid sequences were found to be similar to thecorresponding (i.e., carboxyl-terminal) region of the nucleic acidsequence of human prostatic acid phosphatase. The most highly conservedregion of continuous similarity between flea and human amino acidsequences spans from about amino acid 272 through about amino acid 333of the human enzyme. Comparison of the nucleic acid sequence encodingamino acids from about 268 through about 333 of the human enzyme withthe corresponding regions of nfspN(A) and nfspN(B) nucleic acidsequences indicate that nfspN(A) is about 40% identical, and nfspN(B) isabout 43% identical, to that region of the human prostatic acidphosphatase gene. Comparison of the region spanning from about aminoacid 268 through about amino acid 333 of the human enzyme with thecorresponding regions of PfspN(A) and PfspN(B) indicate that PfspN(A) isabout 28% identical, and PfspN(B) is about 30% identical, to that regionof the human prostatic acid phosphatase gene. The possibility that atleast some fspN proteins encode an active acid phosphatase is supportedby the finding that flea saliva extract FS-3 has been shown to have acidphosphatase activity, as described in Example 3.

[0411] The apparent complete nucleic acid sequence of the coding regionof nucleic acid molecule nfspN(A), referred to herein as nfspN(A)₁₁₉₇,is denoted herein as SEQ ID NO: 55. Translation of SEQ ID NO: 55 yieldsan apparent full-length fspN protein named PfspN(A)₃₉₈ having an aminoacid sequence denoted herein as SEQ ID NO: 56. (It should be noted thatalthough nucleic acid sequence SEQ ID NO: 55 and amino acid sequence SEQID NO: 56 do not exactly match nucleic acid sequence SEQ ID NO: 50 oramino acid sequences SEQ ID NO: 51 or SEQ ID NO: 54 in the correspondingregions, the mismatches are likely due to sequencing errors in SEQ IDNO: 50, SEQ ID NO: 51 and SEQ ID NO: 54.)

[0412] Comparison of SEQ ID NO: 56 with the N-terminal amino acidsequences obtained for fspN1 (SEQ ID NO: 11), fspN2 (SEQ ID NO: 12) andfspN3 (SEQ ID NO: 13) indicates that the amino terminal amino acids offspN1 and fspN2 correspond to amino acid position 18 of SEQ ID NO: 56,while the amino terminal amino acid of fspN3 corresponds to amino acidposition 20 of SEQ ID NO: 56. SEQ ID NO: 13 appears to be identical tothe region of SEQ ID NO: 56 spanning amino acid positions from 20through 39, suggesting that nfspN(A) encodes fspN3. SEQ ID NO: 11 isabout 67% identical to the corresponding region of SEQ ID NO: 56, andSEQ ID NO: 12 is about 60% identical (discounting the 3 unknown aminoacids in SEQ ID NO: 12) to the corresponding region of SEQ ID NO: 56,supporting the suggestion that fspN1 and fspN2 are members of the sameflea saliva protein family as fspN3.

[0413] Comparison of SEQ ID NO: 56 with the amino acid sequence of humanprostatic acid phosphatase indicates that the two sequences share about30% identity at the amino acid level.

Example 13

[0414] This Example demonstrates the production of a bacterialrecombinant cell including an fspN protein and use of that cell toproduce the fspN protein.

[0415] An about 1000 bp DNA fragment, denoted nfspN₁₀₀₀, was PCRamplified from a nucleic acid molecule encoding an fspN protein usingthe following primers: F7 sense, having nucleic acid sequence 5′GGCGTCTCGAGAGAATTGAAATTTGTGTTTGCG 3′ (XhoI site in bold), denoted SEQ IDNO: 46; and F7 antisense, having nucleic acid sequence 5′AGACGAGAATTCCAATTTATCATGAGCGG 3′ (EcoRI site in bold), denoted SEQ IDNO: 47. The PCR product was digested with XhoI and EcoRI restrictionendonucleases, gel purified and subcloned into expression vectorpTrcHisB (available from InVitrogen, Corp.) that had been digested withXhoI and EcoRI to form recombinant molecule pHis-nfspN₁₀₀₀. Therecombinant molecule was transformed into E. coli BL21 competent cells(available from Novagen) to form recombinant cell E.coli:pHis-nfspN₁₀₀₀.

[0416] Recombinant cell E. coli:pHis-nfspN₁₀₀₀ was cultured and inducedas described in Example 11A to produce fusion protein PHIS-PfspN3. Therecombinant fusion protein was detected by immunoblot analysis using theT7 Tag monoclonal antibody as described in Example 11A. PHIS-PfspN3 wasNi purified using Ni-NTA spin kit (available from Qiagen, Chatsworth,Calif.) and the purification verified using T7 Tag monoclonal antibodyas described above.

Example 14

[0417] The Example demonstrates the expression of an fspN protein ininsect cells.

[0418] Recombinant molecule pVL-nfspN₁₀₀₀ containing the nfspN₁₀₀₀nucleic acid molecule operatively linked to baculovirus polyhedrintranscription control sequences was produced in the following manner. Anabout 1000 bp DNA fragment, denoted nfspN₁₀₀₀, was PCR amplified from anucleic acid molecule encoding an fspN protein using the followingprimers: sense primer 17, having nucleic acid sequence 5′ CCG GAA TTCCGG TAT AAA TAT GTG GCG TCT ACT G 3′ (EcoRI site in bold), denoted SEQID NO: 48, and designed to enhance expression in insect cells; andantisense primer 18, having nucleic acid sequence 5′ CCG GAA TTC TTA AGACGA TTT ACA CAA TTT ATC 3′ (EcoRI site in bold), denoted SEQ ID NO: 49.The PCR product was digested with EcoRI and non-directionally clonedinto the baculovirus shuttle vector pVL1393 (available from InVitrogen,Corp.). Orientation was determined by restriction digest with the enzymeEcoRV. The resultant recombinant molecule, i.e., pVL-nfspN₁₀₀₀, wasco-transfected into S. frugiperda cells (donated by the ColoradoBioprocessing Center, Fort Collins, Colo.) with wild type linearbaculovirus DNA (AcMNPV) and insectin cationic liposome according tomanufacturer's specifications (available from InVitrogen Corp.) toproduce recombinant cell S. frugiperda:pVL-nfspN₁₀₀₀. The supernatantwas tested five days post-transfection by Western blot analysis usingrabbit antiserum against flea fspN proteins (as described in Example 12;denoted B2237) and a protein approximately 40 kD was detected. Therecombinant virus, vBV-nfspN₁₀₀₀ was recovered from the supernatant andplaque purified.

Example 15

[0419] This Example demonstrates that use of ELISAs to detect anti-fleasaliva IgE antibodies in the sera of dogs sensitized to fleas or fleasaliva.

[0420] A. In a first study, sera collected from three dogs that had beenartificially sensitized to flea bites were pooled and pretreated bycontacting the pooled sera with Protein G to remove at least some of thenon-IgE immunoglobulins present in the sera. IgE antibodies were thenaffinity-purified from the pretreated sera using Con-A chromatography.

[0421] The affinity-purified IgE antibodies were exposed to thefollowing flea saliva products and proteins: FS-1 saliva extract at 2mg/ml (23,300 flea-hours per μl); fspA, fspB, fspC1, fspC2, fspD1,fspD2, fspE, fspF, fspG, fspH, fspI, fspJ, fspK, fspL, fspM1, fspM2, andfspN (from a 233,000 flea-hours per μl sample applied to HPLCchromatography as described in Example 3). The flea saliva products andproteins were suspended in 0.1 M sodium carbonate, pH 9.6, and 100 μlsamples of each were placed in microtiter dish wells. The samples wereincubated overnight at room temperature, washed 5 times with PBS/Tween,blocked with a solution of PBS, 2% BSA, 0.02% NaN₃, for 1 hour at 37°C., and washed 5 times with PBS/Tween. The washed wells were eachexposed to 100 μl aliquots of the affinity-purified dog IgE antibodiesfor 1 hour at 37° C. The wells were washed 5 times with PBS/Tween andexposed for 1 hour, at 37° C., to 100 μl of a monoclonal mouseanti-canine IgE antibody preparation diluted 1:000 in PBS, 2% BSA, 0.05%Triton X-100. The wells were washed 5 times with PBS/Tween, exposed for1 hour, at 37° C., to 100 μl donkey anti-mouse IgG (H+L)-HRP, and washed5 times with PBS/Tween. The wells were developed with 100 μl KPLTMB:H₂O₂, 1:1, for 10 minutes, the reaction being stopped with 50 μl 2.5N hydrogen sulfate. The wells were read at 450 nm.

[0422] The results, shown in Table 18 and FIG. 10, indicate that FAD+dogs have in their sera IgE antibodies that react in a sensitive andspecific manner with FS-1 flea saliva extract as well as with fleasaliva proteins fspE, fspF, fspG, fspH, fspI, fspJ, fspK, fspL, fspM1,fspM2 and fspN. The IgE antibody preparation reacted minimally, if atall, with flea saliva proteins fspA, fspB, fspC1, fspC2, fspD1 andfspD2. Thus, the IgE reactivity closely followed the skin test resultsof Example 8 in the artificially sensitized dogs with the same fleasaliva products and proteins. TABLE 18 Volume of Antigen Fraction 0.5 μl0.25 μl 0.125 μl 0.063 μl A 0.007 0.008 0.012 0.018 B 0.016 0.010 0.0130.052 C1 0.035 0.008 0.035 0.020 C2 0.022 0.009 0.002 0.005 D1 0.0130.025 0.004 0.005 D2 0.059 0.018 0.017 0.012 E 0.214 0.263 0.206 0.092 F0.276 0.393 0.217 0.114 G 0.288 0.217 −0.010 −0.010 H 0.503 0.336 0.2030.062 I 1.076 0.997 0.917 0.637 J 0.955 0.816 0.673 0.456 K 1.095 0.8980.815 0.690 L 0.991 0.721 0.485 0.162 M1 1.251 1.190 0.840 0.454 M21.561 1.105 0.902 0.558 N 1.989 1.887 1.819 1.435 FS-1 1.367 1.246 0.9820.604 none 0.002 0.005 0.008 0.121

[0423] B. In a second study, serum collected from a dog that had beenartificially sensitized to flea bites was pretreated by contacting theserum with Protein G to remove at least some of the non-IgEimmunoglobulins present in the serum. The reactivity of the pretreatedserum to FS-1 flea saliva extract was determined as described in Example15A. Also tested was the reactivity to FS-1 flea saliva extract of seracollected from dogs infected with heartworm, pooled and pretreated bycontacting the serum with Protein G. The results, shown in Table 19 andFIGS. 11A and 11B, demonstrate a dose dependent reactivity of IgE fromthe FAD+ dog while IgE from heartworm infected dogs had no reactivityagainst FS-1 flea saliva extract. TABLE 19 Sera dil. 1:2 1:4 1:8 1:161:32 1:64 1:128 none DOG 2082128   2 μg 1.67 1.20 0.85 0.57 0.34 0.190.11 0.01   1 μg 1.43 1.16 0.80 0.49 0.30 0.17 0.10 0.00  0.5 μg 1.321.02 0.71 0.46 0.28 0.14 0.08 0.00 0.25 μg 1.18 0.92 0.59 0.38 0.22 0.120.06 0.00 0.13 μg 0.95 0.80 0.52 0.30 0.19 0.11 0.06 0.00 none 0.00 0.010.00 0.00 0.00 0.00 0.00 0.01 HEARTWORM POOL   2 μg 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00   1 μg 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00  0.5μg 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.25 μg 0.00 0.01 0.01 0.000.00 0.00 0.00 0.00 0.13 μg 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 none0.01 0.01 0.01 0.00 0.00 0.00 0.01 0.00

Example 16

[0424] This example demonstrates the ability of formulations of thepresent invention, including formulations comprising flea saliva extractFS-1, E. coli-produced fspH, E. coli-produced fspN3 or insect cell S.frugiperda-produced fspN3 to identify animals susceptible to fleaallergy dermatitis (i.e., to induce flea allergy dermatitis in an animalsusceptible to flea allergy dermatitis).

[0425] The formulations were produced as follows. FS-1 was produced asdescribed in Example 2. E. coli-produced fspH was produced by an E. colicell transformed with a nucleic acid molecule encoding fspH operativelylinked to expression vector λP_(R)/T²ori/S10HIS-RSET-A9, the productionof which is described in PCT/US95/02941, Example 7; the resultantPHIS-fspH fusion protein was purified as described in Example 13. E.coli-produced fspN3 and S. frugiperda-produced fspN3 were produced asdescribed in Examples 13 and 14, respectively; E. coli-produced fspN3was purified as described in Example 11A; S. frugiperda-produced fspN3was purified by anion/cation exchange chromatography.

[0426] The formulations were tested in artificially sensitized dogs2080109, 2082101, 2082128, BFQ2, CPO2, CQQ2, as described in Example 8.The injected samples were as follows: (a) saline negative control; (b)histamine positive control; (c) 2 μg FS-1; (d) 0.1 μg E. coli-producedfspH; (e) 1.0 μg E. coli-produced fspH; (f) 0.2 μg E. coli-producedfspN3; (g) 2.0 μg E. coli-produced fspN3; (h) 0.2 μg S.frugiperda-produced fspN3; and (i) 2.0 μg S. frugiperda-produced fspN3.The immediate hypersensitivity results are shown in Table 20, and thedelayed hypersensitivity results are shown in Table 21. Scoring was asdescribed in Example 8; NA indicates a bad injection. TABLE 20 a)Immediate (15 min.) subjective score (1-4+) (NA was a bad injection) E.coli E. coli S. frugiperda FS-H fspN3 fspN3 Dog Saline Histamine FS-10.1 1.0 0.2 2.0 0.2 2.0 109 0 4 4 NA 4 1 1 1 1 101 0 4 4 4 4 1 2 0 1 1280 4 4 2 4 1 2 1 3 BFQ2 0 4 3 0 3 0 0 0 2 CPO2 0 4 4 4 4 1 4 1 2 CQQ2 0 44 4 4 0 0 0 2

[0427] TABLE 21 b) Delayed (24 hr.) subjective score (1-4+) (NA was abad injection) E. coli E. coli S. frugiperda FS-H fspN3 fspN3 Dog SalineHistamine FS-1 0.1 1.0 0.2 2.0 0.2 2.0 109 0 0 0 NA 0 0 0 0 0 101 0 0 30 2 2 3 2 3 128 0 0 3 0 2 2 2 2 3 BFQ2 0 0 1 0 0 0 0 0 0 CPO2 0 0 0 0 00 0 0 0 CQQ2 0 0 0 0 0 0 0 0 0

[0428] In summary, these results indicate that E. coli-produced fspHexhibited a strong positive immediate reaction in all dogs, the reactionbeing proportional to the dogs' reaction to flea saliva. E.coli-produced and S. frugiperda-produced fspN3 proteins also exhibited apositive immediate reaction in 4 and all dogs, respectively. The twodogs that showed a strong delayed hypersensitive response reaction toFS-1 showed similar delayed hypersensitive response reactions torecombinantly produced fspH and fspN3.

[0429] Sequence Listing

[0430] The following Sequence Listing is submitted pursuant to 37 CFR§1.821. A copy in computer readable form is also submitted herewith.

[0431] Applicants assert pursuant to 37 CFR §1.821 that the content ofthe paper and computer readable copies of SEQ ID NO: 1 through SEQ IDNO: 54 submitted herewith are the same.

1 56 22 amino acids amino acid linear protein Xaa = Tyr, Gln, Ser or Arg1 Xaa = Gly or Tyr 2 Xaa = Lys or Ser 3 Xaa = Gly or Ser 9 Xaa = Gly orLys 10 Xaa = Arg or Ile 14 Xaa = Ile or Leu 17 Xaa = Lys or Asp 19 Xaa =Gly or Leu 21 1 Xaa Xaa Xaa Gln Tyr Ser Glu Lys Xaa Xaa Arg Gly Gln XaaHis Gln 1 5 10 15 Xaa Leu Xaa Lys Xaa Lys 20 78 amino acids amino acidlinear protein Xaa = His or Tyr 27 2 Asp Arg Arg Val Ser Lys Thr Cys GlnSer Gly Gly Lys Ile Gln Ser 1 5 10 15 Glu Xaa Gln Val Val Ile Lys SerGly Gln Xaa Ile Leu Glu Asn Tyr 20 25 30 Xaa Ser Asp Gly Arg Asn Asn AsnAsn Pro Cys His Leu Phe Cys Met 35 40 45 Arg Glu Cys Arg Ser Gly Asn GlyGly Cys Gly Asn Gly Gly Arg Thr 50 55 60 Arg Pro Asp Ser Lys His Cys TyrCys Glu Ala Pro Tyr Ser 65 70 75 12 amino acids amino acid linearprotein 3 Asp Ser Lys His Cys Tyr Cys Glu Ala Pro Tyr Ser 1 5 10 37amino acids amino acid linear protein 4 Asp Gly Arg Asn Asn Asn Asn ProCys His Leu Phe Cys Met Arg Glu 1 5 10 15 Cys Arg Ser Gly Asn Gly GlyCys Gly Asn Gly Gly Arg Thr Arg Pro 20 25 30 Asp Ser Lys His Cys 35 11amino acids amino acid linear protein 5 Asp Arg Arg Val Ser Lys Thr CysGln Ser Gly 1 5 10 38 amino acids amino acid linear protein 6 Glu AspIle Trp Lys Val Asn Lys Lys Xaa Thr Ser Gly Gly Lys Asn 1 5 10 15 GlnAsp Arg Lys Leu Asp Gln Ile Ile Gln Lys Gly Gln Gln Val Xaa 20 25 30 XaaGln Asn Xaa Xaa Lys 35 23 amino acids amino acid linear protein 7 AsnSer His Glu Pro Gly Asn Thr Arg Lys Ile Arg Glu Val Met Asp 1 5 10 15Lys Leu Arg Lys Gln His Pro 20 27 amino acids amino acid linear protein8 Glu Ile Lys Arg Asn Ser His Glu Pro Gly Asn Thr Arg Lys Ile Arg 1 5 1015 Glu Val Met Asp Lys Leu Arg Lys Gln His Pro 20 25 36 amino acidsamino acid linear protein 9 Asn Asp Lys Glu Pro Gly Asn Thr Arg Lys IleArg Glu Val Met Asp 1 5 10 15 Lys Leu Arg Lys Gln Ala Gln Pro Arg ThrAsp Gly Gln Arg Pro Lys 20 25 30 Thr Xaa Ile Met 35 21 amino acids aminoacid linear protein 10 Xaa Leu Xaa Arg Asn Asp Lys Glu Pro Gly Asn ThrArg Lys Ile Arg 1 5 10 15 Glu Val Met Asp Lys 20 12 amino acids aminoacid linear DNA (genomic) 11 Asn Asp Glu Leu Lys Phe Val Phe Val Met AlaLys 1 5 10 23 amino acids amino acid linear protein 12 Xaa Asp Glu LeuLys Phe Val Phe Val Met Ala Lys Gly Pro Ser Xaa 1 5 10 15 Gln Ala XaaAsp Tyr Pro Cys 20 20 amino acids amino acid linear protein 13 Glu LeuLys Phe Val Phe Ala Thr Ala Arg Gly Met Ser His Thr Pro 1 5 10 15 CysAsp Tyr Pro 20 37 amino acids amino acid linear protein Xaa = His or Tyr27 14 Asp Arg Arg Val Ser Lys Thr Xaa Gln Ser Gly Gly Lys Ile Gln Ser 15 10 15 Glu Xaa Gln Val Val Ile Lys Ser Gly Gln Xaa Ile Leu Glu Asn Tyr20 25 30 Xaa Ser Asp Gly Arg 35 20 base pairs nucleic acid single linearDNA (primer) 15 AAYAAYAAYA AYCCNTGYCA 20 17 base pairs nucleic acidsingle linear DNA (primer) 16 CAYTCNCKCA TRCARAA 17 18 base pairsnucleic acid single linear DNA (primer) 17 TGYCAYYTNT TYTGYATG 18 23base pairs nucleic acid single linear DNA (primer) 18 GGNGCYTCRCARTARCARTG YTT 23 17 base pairs nucleic acid single linear DNA (primer)19 GTAAAACGAC GGCCAGT 17 93 base pairs nucleic acid single linear cDNA20 TTGTCACTTT TTTTGTATGA GAGAATGCAG GTCAGGAAAC GGCGGTTGCG GAAACGGAGG 60AAGGACAAGA CCTGATTCGA AGCACTGCTA TGC 93 23 base pairs nucleic acidsingle linear DNA (primer) 21 GARGAYATHT GGAARGTNAA YAA 23 23 base pairsnucleic acid single linear DNA (primer) 22 ACNTCNGGNG GNAARAAYCA RGA 2320 base pairs nucleic acid single linear DNA (primer) 23 TTGGGTACCGGGCCCCCCCT 20 573 base pairs nucleic acid single linear cDNA 24CTTACGTCCG GGGGTAAGAA TCAGGATAGA AAACTCGATC AAATAATTCA AAAAGGCCAA 60CAAGTTAAAA TCCAAAATAT TTGCAAATTA ATACGAGATA AACCACATAC AAATCAAGAG 120AAAGAAAAAT GTATGAAATT TTGCACAAAA AACGTTTGCA AAGGTTATAG AGGAGCTTGT 180GATGGCAATA TTTGCTACTG CAGCAGGCCA AGTAATTTAG GTCCTGATTG GAAAGTCAAC 240GAAAGAATCG AAAGACTCCC AATAACAAAG ATTCTCGTCT CAGGAAATAG TTCCATATCG 300ACAACAATTA CGAATTCCAA ATATTTCGAA ACTAAAAATT CAGAGACCAA TGAAGATTCC 360AAATCGAAAA AACATTCGAA AGAAAAATGT CGTGGTGGAA ATGATGCTGG ATGTGATGGA 420AACGTTTTGT TATTGTCGAC CAAAAAATAA ATAATAATTA TAATAAATAA ATTGTTATAG 480TTATTAGTTA TCCCGTCACA TATTAGAAAA GTGGCTTATA ATTTATGAAC AATATAACAC 540ATAAATTAGT TGTGTAAAAA AAAAAAAAAA AAA 573 149 amino acids amino acidlinear protein 25 Leu Thr Ser Gly Gly Lys Asn Gln Asp Arg Lys Leu AspGln Ile Ile 1 5 10 15 Gln Lys Gly Gln Gln Val Lys Ile Gln Asn Ile CysLys Leu Ile Arg 20 25 30 Asp Lys Pro His Thr Asn Gln Glu Lys Glu Lys CysMet Lys Phe Cys 35 40 45 Thr Lys Asn Val Cys Lys Gly Tyr Arg Gly Ala CysAsp Gly Asn Ile 50 55 60 Cys Tyr Cys Ser Arg Pro Ser Asn Leu Gly Pro AspTrp Lys Val Asn 65 70 75 80 Glu Arg Ile Glu Arg Leu Pro Ile Thr Lys IleLeu Val Ser Gly Asn 85 90 95 Ser Ser Ile Ser Thr Thr Ile Thr Asn Ser LysTyr Phe Glu Thr Lys 100 105 110 Asn Ser Glu Thr Asn Glu Asp Ser Lys SerLys Lys His Ser Lys Glu 115 120 125 Lys Cys Arg Gly Gly Asn Asp Arg GlyCys Asp Gly Asn Val Leu Leu 130 135 140 Leu Ser Thr Lys Lys 145 158amino acids amino acid linear protein 26 Glu Asp Ile Trp Lys Val Asn LysLys Leu Thr Ser Gly Gly Lys Asn 1 5 10 15 Gln Asp Arg Lys Leu Asp GlnIle Ile Gln Lys Gly Gln Gln Val Lys 20 25 30 Ile Gln Asn Ile Cys Lys LeuIle Arg Asp Lys Pro His Thr Asn Gln 35 40 45 Glu Lys Glu Lys Cys Met LysPhe Cys Thr Lys Asn Val Cys Lys Gly 50 55 60 Tyr Arg Gly Ala Cys Asp GlyAsn Ile Cys Tyr Cys Ser Arg Pro Ser 65 70 75 80 Asn Leu Gly Pro Asp TrpLys Val Asn Glu Arg Ile Glu Arg Leu Pro 85 90 95 Ile Thr Lys Ile Leu ValSer Gly Asn Ser Ser Ile Ser Thr Thr Ile 100 105 110 Thr Asn Ser Lys TyrPhe Glu Thr Lys Asn Ser Glu Thr Asn Glu Asp 115 120 125 Ser Lys Ser LysLys His Ser Lys Glu Lys Cys Arg Gly Gly Asn Asp 130 135 140 Arg Gly CysAsp Gly Asn Val Leu Leu Leu Ser Thr Lys Lys 145 150 155 10 amino acidsamino acid linear protein Xaa = Ser or Gln 1 Xaa = any amino acid 8 Xaa= Gly or Lys 9 27 Xaa Gly Lys Gln Tyr Ser Glu Xaa Xaa Lys 1 5 10 6 aminoacids amino acid linear protein 28 Asp Arg Arg Val Ser Lys 1 5 23 aminoacids amino acid linear protein Xaa = any amino acid 8 29 Ser Lys MetVal Thr Glu Lys Xaa Lys Ser Gly Gly Asn Asn Pro Ser 1 5 10 15 Thr LysGlu Val Ser Ile Pro 20 20 amino acids amino acid linear protein Xaa =any amino acid 15 30 Glu Val Ser Ile Pro Ser Gly Lys Leu Thr Ile Glu AspPhe Xaa Ile 1 5 10 15 Gly Asn His Gln 20 25 amino acids amino acidlinear protein 31 Asp Ile Glu Asn Ile Lys Lys Gly Glu Gly Gln Pro GlyAla Pro Gly 1 5 10 15 Gly Lys Glu Asn Asn Leu Ser Val Leu 20 25 242 basepairs nucleic acid single linear cDNA CDS 3..242 32 AT TTT TCC CTT TGTGTT TTA TAC CAA ATT GTG GTT GCT GAT AGA AGA 47 Phe Ser Leu Cys Val LeuTyr Gln Ile Val Val Ala Asp Arg Arg 1 5 10 15 GTT TCC AAA ACA TGT CAAAGT GGA GGA AAG ATA CAA AGT GAG GAG CAA 95 Val Ser Lys Thr Cys Gln SerGly Gly Lys Ile Gln Ser Glu Glu Gln 20 25 30 GTG GTA ATT AAA TCT GGA CAACAT ATT CTT GAA AAT TAT TGC TCA GAT 143 Val Val Ile Lys Ser Gly Gln HisIle Leu Glu Asn Tyr Cys Ser Asp 35 40 45 GGG AGA AAT AAT AAT AAT CCA TGCCAC TTG TTT TGT ATG AGA GAA TGC 191 Gly Arg Asn Asn Asn Asn Pro Cys HisLeu Phe Cys Met Arg Glu Cys 50 55 60 AGG TCA GGA AAC GGC GGT TGC GGA AACGGA GGA AGG ACA AGA CCT GAT 239 Arg Ser Gly Asn Gly Gly Cys Gly Asn GlyGly Arg Thr Arg Pro Asp 65 70 75 TCG 242 Ser 80 80 amino acids aminoacid linear protein 33 Phe Ser Leu Cys Val Leu Tyr Gln Ile Val Val AlaAsp Arg Arg Val 1 5 10 15 Ser Lys Thr Cys Gln Ser Gly Gly Lys Ile GlnSer Glu Glu Gln Val 20 25 30 Val Ile Lys Ser Gly Gln His Ile Leu Glu AsnTyr Cys Ser Asp Gly 35 40 45 Arg Asn Asn Asn Asn Pro Cys His Leu Phe CysMet Arg Glu Cys Arg 50 55 60 Ser Gly Asn Gly Gly Cys Gly Asn Gly Gly ArgThr Arg Pro Asp Ser 65 70 75 80 591 base pairs nucleic acid singlelinear cDNA CDS 1..466 34 TGG AAA GTT AAT AAA AAA TGT ACA TCA GGT GGAAAA AAT CAA GAT AGA 48 Trp Lys Val Asn Lys Lys Cys Thr Ser Gly Gly LysAsn Gln Asp Arg 1 5 10 15 AAA CTC GAT CAA ATA ATT CAA AAA GGC CAA CAAGTT AAA ATC CAA AAT 96 Lys Leu Asp Gln Ile Ile Gln Lys Gly Gln Gln ValLys Ile Gln Asn 20 25 30 ATT TGC AAA TTA ATA CGA GAT AAA CCA CAT ACA AATCAA GAG AAA GAA 144 Ile Cys Lys Leu Ile Arg Asp Lys Pro His Thr Asn GlnGlu Lys Glu 35 40 45 AAA TGT ATG AAA TTT TGC ACA AAA AAC GTT TGC AAA GGTTAT AGA GGA 192 Lys Cys Met Lys Phe Cys Thr Lys Asn Val Cys Lys Gly TyrArg Gly 50 55 60 GCT TGT GAT GGC AAT ATT TGC TAC TGC AGC AGG CCA AGT AATTTA GGT 240 Ala Cys Asp Gly Asn Ile Cys Tyr Cys Ser Arg Pro Ser Asn LeuGly 65 70 75 80 CCT GAT TGG AAA GTC AAC GAA AGA ATC GAA AGA CTC CCA ATAACA AAG 288 Pro Asp Trp Lys Val Asn Glu Arg Ile Glu Arg Leu Pro Ile ThrLys 85 90 95 ATT CTC GTC TCA GGA AAT AGT TCC ATA TCG ACA ACA ATT ACG AATTCC 336 Ile Leu Val Ser Gly Asn Ser Ser Ile Ser Thr Thr Ile Thr Asn Ser100 105 110 AAA TAT TTC GAA ACT AAA AAT TCA GAG ACC AAT GAA GAT TCC AAATCG 384 Lys Tyr Phe Glu Thr Lys Asn Ser Glu Thr Asn Glu Asp Ser Lys Ser115 120 125 AAA AAA CAT TCG AAA GAA AAA TGT CGT GGT GGA AAT GAT CGT GGATGT 432 Lys Lys His Ser Lys Glu Lys Cys Arg Gly Gly Asn Asp Arg Gly Cys130 135 140 GAT GGA AAC GTT TTG TTA TTG TCG ACC AAA AAA T AAATAATAAT 476Asp Gly Asn Val Leu Leu Leu Ser Thr Lys Lys 145 150 155 TATAATAAATAAATTGTTAT AGTTATTAGT TATCCCGTCA CATATTAGAA AAGTGGCTTA 536 TAATTTATGAACAATATAAC ACATAAATTA GTTGTGTAAA AAAAAAAAAA AAAAA 591 155 amino acidsamino acid linear protein 35 Trp Lys Val Asn Lys Lys Cys Thr Ser Gly GlyLys Asn Gln Asp Arg 1 5 10 15 Lys Leu Asp Gln Ile Ile Gln Lys Gly GlnGln Val Lys Ile Gln Asn 20 25 30 Ile Cys Lys Leu Ile Arg Asp Lys Pro HisThr Asn Gln Glu Lys Glu 35 40 45 Lys Cys Met Lys Phe Cys Thr Lys Asn ValCys Lys Gly Tyr Arg Gly 50 55 60 Ala Cys Asp Gly Asn Ile Cys Tyr Cys SerArg Pro Ser Asn Leu Gly 65 70 75 80 Pro Asp Trp Lys Val Asn Glu Arg IleGlu Arg Leu Pro Ile Thr Lys 85 90 95 Ile Leu Val Ser Gly Asn Ser Ser IleSer Thr Thr Ile Thr Asn Ser 100 105 110 Lys Tyr Phe Glu Thr Lys Asn SerGlu Thr Asn Glu Asp Ser Lys Ser 115 120 125 Lys Lys His Ser Lys Glu LysCys Arg Gly Gly Asn Asp Arg Gly Cys 130 135 140 Asp Gly Asn Val Leu LeuLeu Ser Thr Lys Lys 145 150 155 34 base pairs nucleic acid single linearprimer 36 ATTCGGATCC ATGGAAAGTT AATAAAAAAT GTAC 34 32 base pairs nucleicacid single linear primer 37 TAATGGATCC TTATTTTTTG GTCGACAATA AC 32 22base pairs nucleic acid single linear primer 38 CCTGACCTGC ATTCTCTCAT AC22 27 base pairs nucleic acid single linear primer 39 AGGTCTTGTCCTTCCTCCGT TTCCGCA 27 19 base pairs nucleic acid single linear primer 40GGAAACAGCT ATGACCATG 19 17 base pairs nucleic acid single linear primer41 ATTAACCCTC ACTAAAG 17 22 base pairs nucleic acid single linear primer42 GCAAAGGTTA TAGAGGAGCT TG 22 20 base pairs nucleic acid single linearprimer 43 AGCTTTCCAT CACATCCAGC 20 54 base pairs nucleic acid singlelinear primer 44 CGCGGATCCT ATAAATATGG AGGACATCTG GAAAGTTAAT AAAAAATGTACATC 54 39 base pairs nucleic acid single linear primer 45 GCTCTAGAGCATTTATTTTT TGGTCGACAA TAACAAAAC 39 33 base pairs nucleic acid singlelinear primer 46 GGCGTCTCGA GAGAATTGAA ATTTGTGTTT GCG 33 29 base pairsnucleic acid single linear primer 47 AGACGAGAAT TCCAATTTAT CATGAGCGG 2934 base pairs nucleic acid single linear primer 48 CCGGAATTCC GGTATAAATATGTGGCGTCT ACTG 34 33 base pairs nucleic acid single linear primer 49CCGGAATTCT TAAGACGATT TACACAATTT ATC 33 646 base pairs nucleic acidsingle linear cDNA CDS 3..519 50 CG GCA CGA GCT CGG TCT GTT GGA AGT ATGAAA AAC AAA TTG AAA AGT 47 Ala Arg Ala Arg Ser Val Gly Ser Met Lys AsnLys Leu Lys Ser 1 5 10 15 TTT TCC GAG AAA TAT GTA TGG GCG GCT TTA ACTTCT AAC GAC AAT CTT 95 Phe Ser Glu Lys Tyr Val Trp Ala Ala Leu Thr SerAsn Asp Asn Leu 20 25 30 AGG AAA ATG TCT GGA GGT CGT ATG ATT AAC GAT ATATTG AAC GAT ATC 143 Arg Lys Met Ser Gly Gly Arg Met Ile Asn Asp Ile LeuAsn Asp Ile 35 40 45 GAT AAT ATA AAG AAA GGA AAT GGA CAA CCG AAT GCT CCTGGA AAA ACT 191 Asp Asn Ile Lys Lys Gly Asn Gly Gln Pro Asn Ala Pro GlyLys Thr 50 55 60 GAA AAT AAA TTA TCG GTG TCT GAC CGT TCC TCA AGG TAT CTTAGC AGC 239 Glu Asn Lys Leu Ser Val Ser Asp Arg Ser Ser Arg Tyr Leu SerSer 65 70 75 ATT CGT TTC AGC CTT TTT CGT CCA AGG TAC AAA ATT GAA AAT CAGGAC 287 Ile Arg Phe Ser Leu Phe Arg Pro Arg Tyr Lys Ile Glu Asn Gln Asp80 85 90 95 CTT GAA CCG TCT AGT TTA TAT CCT GGC CAA GGA GCC CTC CAT GTTATT 335 Leu Glu Pro Ser Ser Leu Tyr Pro Gly Gln Gly Ala Leu His Val Ile100 105 110 GAA CTG CAC AAA GAT AAG AAT CAG TGG AAT GTA AAA ACC CTC TATAGA 383 Glu Leu His Lys Asp Lys Asn Gln Trp Asn Val Lys Thr Leu Tyr Arg115 120 125 AAC AAT GAC CAA CAG GAA CTC AAA CCT ATG AAA CTT GCA AAA TGCGGT 431 Asn Asn Asp Gln Gln Glu Leu Lys Pro Met Lys Leu Ala Lys Cys Gly130 135 140 GAC ACA TGT TCT TAT GAA ACT TTC AAA TCA ACT CTA CAA TCC TATAAC 479 Asp Thr Cys Ser Tyr Glu Thr Phe Lys Ser Thr Leu Gln Ser Tyr Asn145 150 155 ATG GAT AAG ACC GCT CAT GAT AAA TTG TGT AAA TCG TCT TAAAAATTATT 529 Met Asp Lys Thr Ala His Asp Lys Leu Cys Lys Ser Ser 160165 170 CGTGAAAAAT AGAATTTTAT TGCTATTTTC TGTAAAACCA TATAAAGCTATTTTAATACT 589 TTGTACAGTA TATACATAAT AAATTGCTAC ATTTGCTCTA AAAAAAAAAAAAAAAAA 646 172 amino acids amino acid linear protein 51 Ala Arg Ala ArgSer Val Gly Ser Met Lys Asn Lys Leu Lys Ser Phe 1 5 10 15 Ser Glu LysTyr Val Trp Ala Ala Leu Thr Ser Asn Asp Asn Leu Arg 20 25 30 Lys Met SerGly Gly Arg Met Ile Asn Asp Ile Leu Asn Asp Ile Asp 35 40 45 Asn Ile LysLys Gly Asn Gly Gln Pro Asn Ala Pro Gly Lys Thr Glu 50 55 60 Asn Lys LeuSer Val Ser Asp Arg Ser Ser Arg Tyr Leu Ser Ser Ile 65 70 75 80 Arg PheSer Leu Phe Arg Pro Arg Tyr Lys Ile Glu Asn Gln Asp Leu 85 90 95 Glu ProSer Ser Leu Tyr Pro Gly Gln Gly Ala Leu His Val Ile Glu 100 105 110 LeuHis Lys Asp Lys Asn Gln Trp Asn Val Lys Thr Leu Tyr Arg Asn 115 120 125Asn Asp Gln Gln Glu Leu Lys Pro Met Lys Leu Ala Lys Cys Gly Asp 130 135140 Thr Cys Ser Tyr Glu Thr Phe Lys Ser Thr Leu Gln Ser Tyr Asn Met 145150 155 160 Asp Lys Thr Ala His Asp Lys Leu Cys Lys Ser Ser 165 170 612base pairs nucleic acid single linear cDNA CDS 2..461 52 C GGC ACG AGGAAA AAC GAG CTG AAA AGT TTT TCC GAA GAA TAT TTA 46 Gly Thr Arg Lys AsnGlu Leu Lys Ser Phe Ser Glu Glu Tyr Leu 1 5 10 15 TGG AGG GCT CTA ACTTCT AAT GAG AAT CTT AGA AAG ATG TCA GGA GGC 94 Trp Arg Ala Leu Thr SerAsn Glu Asn Leu Arg Lys Met Ser Gly Gly 20 25 30 CGT ATG ATT AAC GAT ATATTG AAC GAT ATC GAT AGT ATA AAA GAA GAA 142 Arg Met Ile Asn Asp Ile LeuAsn Asp Ile Asp Ser Ile Lys Glu Glu 35 40 45 AGG GAC AAC CGG GTG CTC CTGGAA AAA CAG GAA ATT AAA TTA TCA ATG 190 Arg Asp Asn Arg Val Leu Leu GluLys Gln Glu Ile Lys Leu Ser Met 50 55 60 CTG ACC GTT CCT CAA GCT ATC TTAGCA GCA TTT GTT TCA GCT TTT GCT 238 Leu Thr Val Pro Gln Ala Ile Leu AlaAla Phe Val Ser Ala Phe Ala 65 70 75 CCC AAA GGT ACA AAA ATT GAA AAT CAGGAC CTT GGT CCG TCT AGT TTA 286 Pro Lys Gly Thr Lys Ile Glu Asn Gln AspLeu Gly Pro Ser Ser Leu 80 85 90 95 TAT CCT GGC CAA GGA GCA CTC CAC GTTATT GAA CTG CAC AAG GAT AAC 334 Tyr Pro Gly Gln Gly Ala Leu His Val IleGlu Leu His Lys Asp Asn 100 105 110 AAC CAA TGG AGT GTG AAA GTT CTC TATAGA AAC AAT GAC AAA ATG GAA 382 Asn Gln Trp Ser Val Lys Val Leu Tyr ArgAsn Asn Asp Lys Met Glu 115 120 125 CTG GAA CCT ATG AAA CTT CCA TCA TGCGAT GAC AAA TGT CCT TGT GAA 430 Leu Glu Pro Met Lys Leu Pro Ser Cys AspAsp Lys Cys Pro Cys Glu 130 135 140 CTT TTA AAT CAA CTC TAC AAT CCT ATGATA T GAAAAAGCAG TCATGTAAAT 481 Leu Leu Asn Gln Leu Tyr Asn Pro Met Ile145 150 TATGTAAAAA GCAATAAAAC TGTTGGCAAA ACATACACTT GAACATATTCTGCAAAATTA 541 TATGACGTTA TTTTTAATAT CACGAAATAA ACTACAAACA AACATATACAAATAAAAAAA 601 AAAAAAAAAA A 612 153 amino acids amino acid linearprotein 53 Gly Thr Arg Lys Asn Glu Leu Lys Ser Phe Ser Glu Glu Tyr LeuTrp 1 5 10 15 Arg Ala Leu Thr Ser Asn Glu Asn Leu Arg Lys Met Ser GlyGly Arg 20 25 30 Met Ile Asn Asp Ile Leu Asn Asp Ile Asp Ser Ile Lys GluGlu Arg 35 40 45 Asp Asn Arg Val Leu Leu Glu Lys Gln Glu Ile Lys Leu SerMet Leu 50 55 60 Thr Val Pro Gln Ala Ile Leu Ala Ala Phe Val Ser Ala PheAla Pro 65 70 75 80 Lys Gly Thr Lys Ile Glu Asn Gln Asp Leu Gly Pro SerSer Leu Tyr 85 90 95 Pro Gly Gln Gly Ala Leu His Val Ile Glu Leu His LysAsp Asn Asn 100 105 110 Gln Trp Ser Val Lys Val Leu Tyr Arg Asn Asn AspLys Met Glu Leu 115 120 125 Glu Pro Met Lys Leu Pro Ser Cys Asp Asp LysCys Pro Cys Glu Leu 130 135 140 Leu Asn Gln Leu Tyr Asn Pro Met Ile 145150 56 amino acids amino acid linear protein 54 Met Trp Arg Leu Leu LeuVal Ile Ser Ser Ala Leu Ile Ile Gln Asn 1 5 10 15 Val Asn Ala Glu LeuLys Phe Val Phe Ala Thr Ala Thr Arg Tyr Val 20 25 30 Ser His Thr Pro SerPro Cys Asp Pro Gly Gly Pro Lys Ile Thr Asn 35 40 45 Lys Pro Gly Asp PheGln Arg Val 50 55 1197 base pairs nucleic acid single linear cDNA CDS1..1196 55 ATG TGG CGT CTA CTG TTA GTT ATT TCA AGT GCA CTT ATC ATT CAAAAT 48 Met Trp Arg Leu Leu Leu Val Ile Ser Ser Ala Leu Ile Ile Gln Asn 15 10 15 GTT AAT GCA GAA TTG AAA TTT GTG TTT GCG ACT GCA CGA GGT ATG TCA96 Val Asn Ala Glu Leu Lys Phe Val Phe Ala Thr Ala Arg Gly Met Ser 20 2530 CAT ACA CCT TGT GAT TAT CCA GGC GGT CCA AAA ATT ACT AAC AAG CCT 144His Thr Pro Cys Asp Tyr Pro Gly Gly Pro Lys Ile Thr Asn Lys Pro 35 40 45GAG ACT TCA AGC GTG TTG ACA ACA GCT GGT AAA AAT GAG GCA CTA GAA 192 GluThr Ser Ser Val Leu Thr Thr Ala Gly Lys Asn Glu Ala Leu Glu 50 55 60 ATTGGC AAA CTA TTA TCT GAC CAT TAC AAA AGT AAT TTA ACA GTT AAG 240 Ile GlyLys Leu Leu Ser Asp His Tyr Lys Ser Asn Leu Thr Val Lys 65 70 75 80 GAATGG GAC TCA AGT AAA AAT TAT TGG ACA TTA GCT AGT AAT ACA AGA 288 Glu TrpAsp Ser Ser Lys Asn Tyr Trp Thr Leu Ala Ser Asn Thr Arg 85 90 95 AGA TCTCAA GAA GGA ACA CTT ATT ATT GGT TCT GGA CTA GAA GGA AAG 336 Arg Ser GlnGlu Gly Thr Leu Ile Ile Gly Ser Gly Leu Glu Gly Lys 100 105 110 AGT AGAGCA GCA GAG TGG TCA CAA GAG ATA GGA AAG AAA ACC ACA TTT 384 Ser Arg AlaAla Glu Trp Ser Gln Glu Ile Gly Lys Lys Thr Thr Phe 115 120 125 TCA GGATTT TCT GAG TAT GCT AAA TTT TAT AGT CAA AAA GAA TGC CCA 432 Ser Gly PheSer Glu Tyr Ala Lys Phe Tyr Ser Gln Lys Glu Cys Pro 130 135 140 AAC TTCATA AAA CAA CAG TTG GAT GCA GTG AAG GAC TTG TTA AAG AGT 480 Asn Phe IleLys Gln Gln Leu Asp Ala Val Lys Asp Leu Leu Lys Ser 145 150 155 160 GCAAAA GAA TAT AAT ACA GAA TTT GAC AAA TTA AAG AAA GTG TAT AAT 528 Ala LysGlu Tyr Asn Thr Glu Phe Asp Lys Leu Lys Lys Val Tyr Asn 165 170 175 ATTGAT GCA ATG AAG GGC CCA CAA AAT GTT TGG CTG GCA TAC GAG ACT 576 Ile AspAla Met Lys Gly Pro Gln Asn Val Trp Leu Ala Tyr Glu Thr 180 185 190 TTAAAT TTA CAA AGC AAG CTC GAT CAG ATT GGT TTG GGA AGT ATG AAA 624 Leu AsnLeu Gln Ser Lys Leu Asp Gln Ile Gly Leu Gly Ser Met Lys 195 200 205 AACAAA TTG AAA AGT TTT TCC GAG AAA TAT GTA TGG GCG GGT TTA ACT 672 Asn LysLeu Lys Ser Phe Ser Glu Lys Tyr Val Trp Ala Gly Leu Thr 210 215 220 TCTAAC GAC AAT CTT AGG AAA ATG TCT GGA GGT CGT ATG ATT AAC GAT 720 Ser AsnAsp Asn Leu Arg Lys Met Ser Gly Gly Arg Met Ile Asn Asp 225 230 235 240ATA TTG AAC GAT ATC GAT AAT ATA AAG AAA GGA AAT GGA CAA CCG AAT 768 IleLeu Asn Asp Ile Asp Asn Ile Lys Lys Gly Asn Gly Gln Pro Asn 245 250 255GCT CCT GGA AAA CTG AAA ATA ATT ATC GGG CTG ACC GCC CCA AGG TTC 816 AlaPro Gly Lys Leu Lys Ile Ile Ile Gly Leu Thr Ala Pro Arg Phe 260 265 270TTA GCA GAA TCC GTT CAG CTT GGG TCC AAG GGT ACA AAA TTG AAT CAG 864 LeuAla Glu Ser Val Gln Leu Gly Ser Lys Gly Thr Lys Leu Asn Gln 275 280 285GAC CAA AAT AAA TTA TCG GTG CTG ACC GTT CCT CAA GGT ATC TTA GCA 912 AspGln Asn Lys Leu Ser Val Leu Thr Val Pro Gln Gly Ile Leu Ala 290 295 300GCA TTC GTT TCA GCT TTT GCT CCC AAA GGT ACA AAA ATT GAA AAT CAG 960 AlaPhe Val Ser Ala Phe Ala Pro Lys Gly Thr Lys Ile Glu Asn Gln 305 310 315320 GAC CTT GAT CCG TCT AGT TTA TAT CCT GGC CAA GGA GCA CTC CAT GTT 1008Asp Leu Asp Pro Ser Ser Leu Tyr Pro Gly Gln Gly Ala Leu His Val 325 330335 ATT GAA CTG CAC AAA GAT AAG AAC CAG TGG AAT GTA AAA ATC CTC TAT 1056Ile Glu Leu His Lys Asp Lys Asn Gln Trp Asn Val Lys Ile Leu Tyr 340 345350 AGA AAC AAT GAC CAA TCG GAA CTC AAA CCT ATG AAA CTT GCA AAA TGC 1104Arg Asn Asn Asp Gln Ser Glu Leu Lys Pro Met Lys Leu Ala Lys Cys 355 360365 GGT GAC ACA TGT TCT TAT GAA ACT TTC AAA TCA ACT CTA CAA TCC TAT 1152Gly Asp Thr Cys Ser Tyr Glu Thr Phe Lys Ser Thr Leu Gln Ser Tyr 370 375380 AAC ATG GAT AAG ACC GCT CAT GAT AAA TTG TGT AAA TCG TCT TAA 1197 AsnMet Asp Lys Thr Ala His Asp Lys Leu Cys Lys Ser Ser 385 390 395 398amino acids amino acid linear protein 56 Met Trp Arg Leu Leu Leu Val IleSer Ser Ala Leu Ile Ile Gln Asn 1 5 10 15 Val Asn Ala Glu Leu Lys PheVal Phe Ala Thr Ala Arg Gly Met Ser 20 25 30 His Thr Pro Cys Asp Tyr ProGly Gly Pro Lys Ile Thr Asn Lys Pro 35 40 45 Glu Thr Ser Ser Val Leu ThrThr Ala Gly Lys Asn Glu Ala Leu Glu 50 55 60 Ile Gly Lys Leu Leu Ser AspHis Tyr Lys Ser Asn Leu Thr Val Lys 65 70 75 80 Glu Trp Asp Ser Ser LysAsn Tyr Trp Thr Leu Ala Ser Asn Thr Arg 85 90 95 Arg Ser Gln Glu Gly ThrLeu Ile Ile Gly Ser Gly Leu Glu Gly Lys 100 105 110 Ser Arg Ala Ala GluTrp Ser Gln Glu Ile Gly Lys Lys Thr Thr Phe 115 120 125 Ser Gly Phe SerGlu Tyr Ala Lys Phe Tyr Ser Gln Lys Glu Cys Pro 130 135 140 Asn Phe IleLys Gln Gln Leu Asp Ala Val Lys Asp Leu Leu Lys Ser 145 150 155 160 AlaLys Glu Tyr Asn Thr Glu Phe Asp Lys Leu Lys Lys Val Tyr Asn 165 170 175Ile Asp Ala Met Lys Gly Pro Gln Asn Val Trp Leu Ala Tyr Glu Thr 180 185190 Leu Asn Leu Gln Ser Lys Leu Asp Gln Ile Gly Leu Gly Ser Met Lys 195200 205 Asn Lys Leu Lys Ser Phe Ser Glu Lys Tyr Val Trp Ala Gly Leu Thr210 215 220 Ser Asn Asp Asn Leu Arg Lys Met Ser Gly Gly Arg Met Ile AsnAsp 225 230 235 240 Ile Leu Asn Asp Ile Asp Asn Ile Lys Lys Gly Asn GlyGln Pro Asn 245 250 255 Ala Pro Gly Lys Leu Lys Ile Ile Ile Gly Leu ThrAla Pro Arg Phe 260 265 270 Leu Ala Glu Ser Val Gln Leu Gly Ser Lys GlyThr Lys Leu Asn Gln 275 280 285 Asp Gln Asn Lys Leu Ser Val Leu Thr ValPro Gln Gly Ile Leu Ala 290 295 300 Ala Phe Val Ser Ala Phe Ala Pro LysGly Thr Lys Ile Glu Asn Gln 305 310 315 320 Asp Leu Asp Pro Ser Ser LeuTyr Pro Gly Gln Gly Ala Leu His Val 325 330 335 Ile Glu Leu His Lys AspLys Asn Gln Trp Asn Val Lys Ile Leu Tyr 340 345 350 Arg Asn Asn Asp GlnSer Glu Leu Lys Pro Met Lys Leu Ala Lys Cys 355 360 365 Gly Asp Thr CysSer Tyr Glu Thr Phe Lys Ser Thr Leu Gln Ser Tyr 370 375 380 Asn Met AspLys Thr Ala His Asp Lys Leu Cys Lys Ser Ser 385 390 395

What is claimed is:
 1. A formulation comprising at least one isolatedectoparasite saliva protein, wherein said ectoparasite saliva proteincomprises at least a portion of an amino acid sequence, wherein saidportion is encoded by a nucleic acid molecule that hybridizes understringent hybridization conditions with a nucleic acid molecule thatencodes a flea saliva protein present in a flea saliva extract selectedfrom the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts.2. A formulation comprising at least one isolated ectoparasite salivaprotein, wherein said ectoparasite saliva protein comprises at least aportion of an amino acid sequence, wherein said portion is encoded by anucleic acid molecule that hybridizes under stringent hybridizationconditions with a nucleic acid molecule that encodes a flea salivaprotein represented as a protein peak in FIG.
 2. 3. A formulationcomprising at least one isolated ectoparasite saliva productsubstantially free of contaminating material, said formulation beingproduced by a process comprising: (a) collecting ectoparasite salivaproducts on a collection means within a saliva collection apparatuscontaining ectoparasites, said apparatus comprising: (i) a housingoperatively connected to a chamber, said chamber having an ambienttemperature warmer than said housing thereby forming a temperaturedifferential between said housing and said chamber, said housing beingcapable of retaining ectoparasites; and (ii) an interface between saidhousing and said chamber, said interface comprising ((a)) a meanscapable of collecting at least a portion of saliva products deposited byectoparasites retained in said apparatus and ((b)) a barrier meanscapable of substantially preventing contaminating material fromcontacting said collection means, wherein said temperature differentialattracts ectoparasites retained in said housing to attempt to feedthrough said barrier means and collection means and, thereby, depositsaliva products on said collection means; and (b) extracting said salivaproducts from said collection means to obtain said formulation.
 4. Aformulation comprising an ectoparasite saliva product, wherein saidformulation, when submitted to Tris glycine SDS-PAGE, comprises afractionation profile as depicted in a figure selected from the groupconsisting of FIG. 1B, lane 13 and FIG. 1B, lane
 14. 5. A therapeuticcomposition for treating allergic dermatitis comprising a formulationcomprising at least one isolated ectoparasite saliva protein, whereinsaid ectoparasite saliva protein comprises at least a portion of anamino acid sequence, wherein said portion is encoded by a nucleic acidmolecule that hybridizes under stringent hybridization conditions with anucleic acid molecule that encodes a flea saliva protein present in aflea saliva extract selected from the group consisting of FS-1, FS-2 andFS-3 flea saliva extracts.
 6. An assay kit for testing if an animal issusceptible to or has allergic dermatitis, said kit comprising: (a) aformulation comprising at least one isolated ectoparasite salivaprotein, wherein said ectoparasite saliva protein comprises at least aportion of an amino acid sequence, wherein said portion is encoded by anucleic acid molecule that hybridizes under stringent hybridizationconditions with a nucleic acid molecule that encodes a flea salivaprotein present in a flea saliva extract selected from the groupconsisting of FS-1, FS-2 and FS-3 flea saliva extracts; and (b) a meansfor determining if said animal is susceptible to or has allergicdermatitis, wherein said means comprises use of said formulation toidentify animals susceptible to or having allergic dermatitis.
 7. Anapparatus to collect ectoparasite saliva products for use in aformulation that are substantially free of contaminating material, saidapparatus comprising: (a) a housing operatively connected to a chamber,said chamber having an ambient temperature warmer than said housingthereby forming a temperature differential between said housing and saidchamber, said housing being capable of retaining ectoparasites; and (b)an interface between said housing and said chamber, said interfacecomprising (i) a means capable of collecting at least a portion ofsaliva products deposited by ectoparasites retained in said apparatusand (ii) a barrier means capable of substantially preventingcontaminating material from contacting said collection means, whereinsaid temperature differential attracts ectoparasites retained in saidhousing to attempt to feed through said barrier means and collectionmeans and, thereby, deposit saliva products on said collection means. 8.A method to produce a formulation comprising ectoparasite salivaproducts, wherein said formulation is substantially free ofcontaminating material, said method comprising: (a) collectingectoparasite saliva products on a collection means within a salivacollection apparatus containing ectoparasites, said apparatuscomprising: (i) a housing operatively connected to a chamber, saidchamber having an ambient temperature warmer than said housing therebyforming a temperature differential between said housing and saidchamber, said housing being capable of retaining ectoparasites; and (ii)an interface between said housing and said chamber, said interfacecomprising ((a)) a means capable of collecting at least a portion ofsaliva products deposited by ectoparasites retained in said apparatusand ((b)) a barrier means capable of substantially preventingcontaminating material from contacting said collection means, whereinsaid temperature differential attracts ectoparasites retained in saidhousing to attempt to feed through said barrier means and collectionmeans and, thereby, deposit saliva products on said collection means;and (b) extracting said products from said collection means to form saidformulation.
 9. A method to identify an animal susceptible to or havingallergic dermatitis, said method comprising: (a) administering to a siteon said animal a formulation comprising at least one isolatedectoparasite saliva protein, wherein said ectoparasite saliva proteincomprises at least a portion of an amino acid sequence, wherein saidportion is encoded by a nucleic acid molecule that hybridizes understringent hybridization conditions with a nucleic acid molecule thatencodes a flea saliva protein present in a flea saliva extract selectedfrom the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts,and administering to a different site on said animal a control solutionselected from the group consisting of positive control solutions andnegative control solutions; and (b) comparing a reaction resulting fromadministration of said formulation with a reaction resulting fromadministration of said control solution, wherein said animal isdetermined to be susceptible to or to have allergic dermatitis if saidreaction to said formulation is at least as large as said reaction tosaid positive control solution, and wherein said animal is determinednot to be susceptible to or not to have allergic dermatitis if saidreaction to said formulation is about the same size as said reaction tosaid negative control solution.
 10. A method to identify an animalsusceptible to or having allergic dermatitis by measuring the presenceof antibodies indicative of allergic dermatitis in said animal, saidmethod comprising: (a) contacting a formulation with a body fluid fromsaid animal under conditions sufficient for formation of animmunocomplex between said formulation and said antibodies, if present,in said body fluid, said formulation comprising at least one isolatedectoparasite saliva protein, wherein said ectoparasite saliva proteincomprises at least a portion of an amino acid sequence, wherein saidportion is encoded by a nucleic acid molecule that hybridizes understringent hybridization conditions with a nucleic acid molecule thatencodes a flea saliva protein present in a flea saliva extract selectedfrom the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts;and (b) determining the amount of immunocomplex formed, whereinformation of said immunocomplex indicates that said animal issusceptible to or has allergic dermatitis.
 11. A method to desensitize ahost animal to allergic dermatitis, comprising administering to saidanimal a therapeutic composition comprising a formulation comprising atleast one isolated ectoparasite saliva protein, wherein saidectoparasite saliva protein comprises at least a portion of an aminoacid sequence, wherein said portion is encoded by a nucleic acidmolecule that hybridizes under stringent hybridization conditions with anucleic acid molecule that encodes a flea saliva protein present in aflea saliva extract selected from the group consisting of FS-1, FS-2 andFS-3 flea saliva extracts.
 12. A method for prescribing treatment forallergic dermatitis, comprising: (a) identifying an animal that issusceptible to or has allergic dermatitis by an in vivo or in vitroassay comprising a formulation comprising at least one isolatedectoparasite saliva protein, wherein said ectoparasite saliva proteincomprises at least a portion of an amino acid sequence, wherein saidportion is encoded by a nucleic acid molecule that hybridizes understringent hybridization conditions with a nucleic acid molecule thatencodes a flea saliva protein present in a flea saliva extract selectedfrom the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts;and (b) prescribing a treatment comprising administering saidformulation to said animal.
 13. The invention of claim 1, 2, 5, 6, 9,10, 11 or 12, wherein said a flea saliva protein is selected from thegroup consisting of fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF,fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2,fspM1, fspM2, fspN1, fspN2 and fspN3.
 14. The invention of claim 1, 2,5, 6, 9, 10, 11 or 12, wherein said flea saliva protein comprises anamino acid sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 54 and SEQ ID NO:
 56. 15. The invention of claim 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 61, 62, 63 or 65, wherein said ectoparasite isselected from the group consisting of fleas, flies, mosquitoes, ticks,mites, lice, spiders, ants and true bugs.
 16. The invention of claim 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 61, 62, 63 or 65, wherein saidectoparasite comprises a flea.
 17. The invention of claim 16, whereinsaid flea is of a species selected from the group consisting ofCtenocephalides canis and Ctenocephalides felis.
 18. The invention ofclaim 1, 2, 3, 4, 5, 6, 9, 10, 11, 12, 61, 62, 63 or 65, wherein saidectoparasite saliva protein or product is selected from the groupconsisting of ectoparasite saliva proteins having molecular weightsranging from about 6 kilodaltons to about 65 kilodaltons as determinedby Tris-glycine SDS-PAGE.
 19. The invention of claim 1, 3, 5, 6, 7, 8,9, 10, 11 or 12, wherein said formulation comprises a flea salivaextract selected from the group consisting of FS-1 flea saliva extract,FS-2 flea saliva extract, FS-3 flea saliva extract and mixtures thereof.20. The invention of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 61,62, 63, 64 or 65, wherein said formulation or a formulation comprisingan expression product of said nucleic acid molecule, comprises at leasta portion of at least one flea saliva protein selected from the groupconsisting of fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1,fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1,fspM2, fspN1, fspN2 and fspN3.
 21. The invention of claim 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12, wherein said formulation comprises anectoparasite saliva protein selected from the group consisting of fspA,fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH,fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2,fspN3, and mixtures thereof.
 22. The invention of claim 1, 2, 4, 5, 6,9, 10, 11 or 12, wherein said formulation is produced by a processcomprising: (a) collecting ectoparasite saliva products on a collectionmeans within a saliva collection apparatus containing ectoparasites,said apparatus comprising: (i) a housing operatively connected to achamber, said chamber having an ambient temperature warmer than saidhousing thereby forming a temperature differential between said housingand said chamber, said housing being capable of retaining ectoparasites;and (ii) an interface between said housing and said chamber, saidinterface comprising ((a)) a means capable of collecting at least aportion of saliva products deposited by ectoparasites retained in saidapparatus and ((b)) a barrier means capable of substantially preventingcontaminating material from contacting said collection means, whereinsaid temperature differential attracts ectoparasites retained in saidhousing to attempt to feed through said barrier means and collectionmeans and, thereby, deposit saliva products on said collection means;and (b) extracting said products from said collection means to form saidformulation.
 23. The invention of claim 22, wherein the ambienttemperature in said chamber ranges from about 20° C. to about 45° C. andthe ambient temperature in said housing ranges from about 5° C. to about35° C.
 24. The invention of claim 22, wherein the relative humidity insaid chamber ranges from about 50% relative humidity to about 100%relative humidity and the relative humidity in said housing ranges fromabout 40% relative humidity to about 60% relative humidity.
 25. Theinvention of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein saidformulation is produced by a process comprising: (a) collectingectoparasite saliva products on a collection means within a salivacollection apparatus containing ectoparasites, said apparatuscomprising: (i) a housing operatively connected to a chamber, saidchamber having an ambient temperature warmer than said housing therebyforming a temperature differential between said housing and saidchamber, said housing being capable of retaining ectoparasites; and (ii)an interface between said housing and said chamber, said interfacecomprising ((a)) a means capable of collecting at least a portion ofsaliva products deposited by ectoparasites retained in said apparatusand ((b)) a barrier means capable of substantially preventingcontaminating material from contacting said collection means, whereinsaid temperature differential attracts ectoparasites retained in saidhousing to attempt to feed through said barrier means and collectionmeans and, thereby, deposit saliva products on said collection means;(b) extracting said products from said collection means with a solutionto form an ectoparasite extract; (c) fractionating said ectoparasiteextract to obtain separated peak fractions; and (d) recovering at leastone of said peak fractions substantially free of the remaining fractionsto obtain said formulation.
 26. The invention of claim 25, whereinseparated peak fractions obtained in step (c) comprise a fractionationprofile as depicted in FIG.
 2. 27. The invention of claim 1, 2, 5, 6, 9,10, 11 or 12, wherein said formulation comprises an ectoparasite salivaprotein produced by a process comprising culturing a recombinant celltransformed with at least one nucleic acid molecule encoding at leastone of said ectoparasite saliva proteins to produce said formulation.28. The invention of claim 1, 2, 3, 4, 5, 6, 7, 8, 61, 62, 63, 64 or 65,wherein said formulation or a formulation comprising an expressionproduct of said nucleic acid molecule, when administered to a hostanimal, is capable of substantially desensitizing said animal toallergic dermatitis.
 29. The invention of claim 28, wherein saidallergic dermatitis is selected from the group consisting of fleaallergy dermatitis, mosquito allergy dermatitis and Culicoides allergydermatitis.
 30. The invention of claim 1, 2, 3, 4, 5, 6, 7, 8, 61, 62,63, 64 or 65, wherein said formulation or a formulation comprising anexpression product of said nucleic acid molecule is used to identifyanimals susceptible to or having allergic dermatitis.
 31. The inventionof claim 30, wherein said allergic dermatitis is selected from the groupconsisting of flea allergy dermatitis, mosquito allergy dermatitis andCulicoides allergy dermatitis.
 32. An isolated antibody capable ofselectively binding to an ectoparasite saliva protein or product as setforth in claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or
 12. 33. Theinvention of claim 1, 2, 4, 5, 6, 9, 10, 11 or 12, wherein saidformulation is substantially free of contaminating material.
 34. Theinvention of claim 33, wherein said contaminating material comprisesmaterial selected from the group consisting of blood proteins, fecalmaterial, larval culture medium, and mixtures thereof.
 35. The inventionof claim 2 or 62, wherein said peak is selected from the groupconsisting of peak A, peak B, peak C, peak D, peak E, peak F, peak G,peak H, peak I, peak J, peak K, peak L, peak M and peak N.
 36. Theinvention of claim 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 61, 62, 63, 64 or65, wherein said formulation or a formulation comprising an expressionproduct of said nucleic acid molecule, when submitted to Tris glycineSDS-PAGE, comprises a fractionation profile as depicted in a figureselected from the group consisting of FIG. 1B, lane 13 and FIG. 1B, lane14.
 37. The invention of claim 1, 2, 3, 5, 6, 7, 8, 9, 10, 11 or 12,wherein said formulation comprises less than about 50 percent ofcontaminating material.
 38. The invention of claim 1, 2, 3, 5, 6, 7, 8,9, 10, 11 or 12, wherein said formulation comprises less than about 10percent of contaminating material.
 39. The invention of claim 5, 6, 9,10, 11, 12 or 63, wherein said composition further comprises at leastone component selected from the group consisting of an excipient, anadjuvant and a carrier.
 40. The invention of claim 5, 11 or 63, whereinsaid composition comprises a controlled release composition.
 41. Theinvention of claim 6, wherein said means of determining is selected fromthe group consisting of in vivo tests and in vitro tests.
 42. Theinvention of claim 41, wherein said in vivo test comprises a skin testcomprising: (a) administering to a site on said animal said formulationand administering to a different site on said animal a control solutionselected from the group consisting of positive control solutions andnegative control solutions; and (b) comparing a reaction resulting fromadministration of said formulation with a reaction resulting fromadministration of said control solution, wherein said animal isdetermined to be susceptible to or to have allergic dermatitis if saidreaction to said formulation is at least as large as said reaction tosaid positive control solution, and wherein said animal is determinednot to be susceptible to or not to have allergic dermatitis if saidreaction to said formulation is about the same size as said reaction tosaid negative control solution.
 43. The invention of claim 6, 9, 10 or12, wherein said kit or said method detects hypersensitivity selectedfrom the group consisting of immediate hypersensitivity and delayedhypersensitivity.
 44. The invention of claim 9 or 42, wherein saidreaction is selected from the group consisting of a wheal, induration,erythema, and combinations thereof.
 45. The invention of claim 41,wherein said in vitro test comprises a method for measuring the presenceof antibodies indicative of allergic dermatitis in said animal, saidmethod comprising: (a) contacting said formulation with a body fluidfrom said animal under conditions sufficient for formation of animmunocomplex between said formulation and said antibodies, if present,in said body fluid; and (b) determining the amount of immunocomplexformed, wherein formation of said immunocomplex indicates that saidanimal is susceptible to or has allergic dermatitis.
 46. The inventionof claim 6 or 10, wherein said formulation is immobilized on asubstrate.
 47. The invention of claim 10 or 45, wherein said antibodiescomprise immunoglobulin IgE antibodies.
 48. The invention of claim 10 or45, wherein said kit detects immediate hypersensitivity in said animal.49. The apparatus of claim 3, 7 or 8, wherein said collection meanscomprises a membrane made of material capable of binding said productsin such a manner that said products can be eluted from said membrane.50. The apparatus of claim 3, 7 or 8, wherein said membrane comprises amaterial selected from the group consisting of polyvinyl difluoride,cellulose esters, nitrocellulose, nylon, polysulfone, andpolytetrafluoroethylene.
 51. The apparatus of claim 3, 7 or 8, whereinsaid membrane comprises a Durapore™ membrane.
 52. The apparatus of claim3, 7 or 8, wherein said membrane comprises a DE-81 chromatography papermembrane.
 53. The apparatus of claim 3, 7 or 8, wherein said barriermeans comprises a material selected from the group consisting ofplastic, teflon, cloth, paper, paraffin and wax.
 54. The apparatus ofclaim 3, 7 or 8, wherein said barrier means comprises Parafilm™.
 55. Theapparatus of claim 3, 7 or 8, wherein said apparatus further comprises ablotting means capable of maintaining a humidity suitable for survivalof said ectoparasite.
 56. An isolated nucleic acid molecule that encodesthe ectoparasite saliva protein of claim 1, 2 or
 3. 57. An isolatednucleic acid molecule that hybridizes under stringent hybridizationconditions with a gene encoding a flea saliva protein present in a fleasaliva extract selected from the group consisting of FS-1, FS-2 and FS-3flea saliva extracts.
 58. A method for producing at least oneectoparasite saliva protein, comprising: (a) culturing a celltransformed with at least one nucleic acid that hybridizes understringent hybridization conditions with a gene encoding a flea salivaprotein present in a flea saliva extract selected from the groupconsisting of FS-1, FS-2 and FS-3 flea saliva extracts to produce saidprotein; and (b) recovering said ectoparasite saliva protein.
 59. Theinvention of claim 56, 57 or 58, wherein said nucleic acid moleculehybridizes under stringent hybridization conditions with a nucleic acidmolecule encoding a flea saliva protein selected from the groupconsisting of fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1,fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1,fspM2, fspN1, fspN2 and fspN3.
 60. The invention of claim 56, 57 or 58,wherein said nucleic acid molecule hybridizes under stringenthybridization conditions with a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 50, SEQ ID NO: 52 and SEQ ID NO:
 55. 61. Arecombinant molecule comprising at least one isolated nucleic acidmolecule as set forth in claim 56, 57 or 58, operatively linked to atleast one transcription control sequence.
 62. A recombinant cellcomprising a cell having at least one isolated nucleic acid molecule asset forth in claim 56, 57 or 58, said cell being capable of expressingsaid nucleic acid molecule.
 63. A therapeutic composition comprising atleast one isolated nucleic acid molecule as set forth in claim 56, 57 or58.
 64. The invention of claim 4, wherein said formulation comprises aflea saliva extract selected from the group consisting of FS-1 fleasaliva extract, FS-2 flea saliva extract and mixtures thereof.
 65. Theinvention of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein saidformulation comprises at least a portion of at least one flea salivaprotein selected from the group consisting of fspE, fspF, fspG1, fspG2,fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2,fspN1, fspN2 and fspN3.
 66. The invention of claim 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or 12, wherein said formulation comprises at least aportion of at least one flea saliva protein selected from the groupconsisting of fspG1, fspG2, fspG3, fspH, fspM1, fspM2, fspN1, fspN2 andfspN3.
 67. The invention of claim 10 or 45, wherein said body fluid ispretreated to remove non-IgE antibodies from said fluid.